Use of Mass Labelled Probes to Detect Target Nucleic Acids Using Mass Spectrometry

ABSTRACT

The invention relates to the use of mass labelled probes to characterise nucleic acids by mass spectrometry. Thus the invention provides methods of detecting the presence of a target nucleic acid in a sample, using a circularising probe in which a mass tag is present in the probe. Further methods of detecting the presence of a target nucleic acid are provided, which in contrast use a probe detection sequence in the circularising probe, wherein the probe detection sequence is detected with a probe attached to a mass tag. Methods for determining a genetic profile from the genome of an organism also form part of the invention.

This invention relates to useful probe molecules for characterisingbiomolecules of interest, particularly nucleic acids. Specifically thisinvention relates to oligonucleotide probes that are cleavably linked totags designed for detection by mass spectrometry and tandem massspectrometry. In addition, this invention relates to associated methodsfor employing mass labeled probes to detect target nucleic acids usingmass spectrometry.

BACKGROUND TO THE INVENTION

Nucleic acids are typically detected by contacting them with labelledprobe molecules under controlled conditions and detecting the labels todetermine whether specific binding or hybridisation has taken place.Various methods of labeling probes are known in the art, including theuse of radioactive atoms, fluorescent dyes, luminescent reagents,electron capture reagents and light absorbing dyes. Each of theselabeling systems has features which make it suitable for certainapplications and not others. For reasons of safety, interest innon-radioactive labeling systems lead to the widespread commercialdevelopment of fluorescent labeling schemes particularly for geneticanalysis. Fluorescent labeling schemes permit the labeling of arelatively small number of molecules simultaneously, typically 4 labelscan be used simultaneously and possibly up to eight. However the costsof the detection apparatus and the difficulties of analysing theresultant signals limit the number of labels that can be usedsimultaneously in a fluorescence detection scheme. More recently therehas been development in the area of mass spectrometry as a method ofdetecting labels that are cleavably attached to their associated probemolecules. Until recently, Mass Spectrometry has been used to detectanalyte ions or their fragment ions directly, however for manyapplications such as nucleic acid analysis, the structure of the analytecan be determined from indirect labeling. This is advantageousparticularly with respect to the use of mass spectrometry becausecomplex biomolecules such as DNA have complex mass spectra and aredetected with relatively poor sensitivity. Indirect detection means thatan associated label molecule can be used to identify the originalanalyte, where the label is designed for sensitive detection and asimple mass spectrum. Simple mass spectra mean that multiple labels canbe used to analyse multiple analytes simultaneously. In fact, many morelabels than can currently be used simultaneously in fluorescence basedassays can be generated.

WO98/31830 describes arrays of nucleic acid probes covalently attachedto cleavable labels that are detectable by mass spectrometry whichidentify the sequence of the covalently linked nucleic acid probe. Thelabeled probes of this application have the structure Nu—L—M where Nu isa nucleic acid covalently linked to L, a cleavable linker, covalentlylinked to M, a mass label. Preferred cleavable linkers in thisapplication cleave within the ion source of the mass spectrometer.Preferred mass labels are substituted poly-aryl ethers. Theseapplication discloses a variety of ionisation methods and analysis byquadrupole mass analysers, TOF analysers and magnetic sector instrumentsas specific methods of analysing mass labels by mass spectrometry.

WO 95/04160 disclose ligands, and specifically nucleic acids, cleavablylinked to mass tag molecules. Preferred cleavable linkers arephoto-cleavable. These application discloses Matrix Assisted LaserDesorption Ionisation (MALDI) Time of Flight (TOF) mass spectrometry asa specific method of analysing mass labels by mass spectrometry.

WO 98/26095 discloses releasable non-volatile mass-label molecules. Inpreferred embodiments these labels comprise polymers, particularlybiopolymers, and more particularly nucleic acids, which are cleavablyattached to a reactive group or ligand, i.e. a probe. Preferredcleavable linkers appear to be chemically or enzymatically cleavable.This application discloses MALDI TOF mass spectrometry as a specificmethod of analysing mass labels by mass spectrometry.

WO 97/27327, WO 97/27325, WO 97/27331 disclose ligands, and specificallynucleic acids, cleavably linked to mass tag molecules. Preferredcleavable linkers appear to be chemically or photo-cleavable. Theseapplication discloses a variety of ionisation methods and analysis byquadrupole mass analysers, TOF analysers and magnetic sector instrumentsas specific methods of analysing mass labels by mass spectrometry.

WO 01/68664 and WO 03/025576 disclose organic molecule mass markers thatare analysed by tandem mass spectrometry. These applications disclosemass markers comprised of two components, a mass tag component and amass normalization component that are connected to each other by acollision cleavable group. Sets of tags can be synthesised where the sumof the masses of the two components produce markers with the sameoverall mass. The mass markers are typically analysed after cleavagefrom their analyte. Analysis takes place in an instrument capable oftandem mass spectrometric analysis. In the first stage of analysis, theMS/MS instrument is set to select ions with the mass-to-charge ratiothat corresponds to the mass marker comprising both the mass tag andmass normaliser, which may be referred to as the ‘parent ion’. Thisselection process effected by the MS/MS instrument allows the markers tobe abstracted from the background. Collision of selected the marker ionsin the second stage of the analysis separates the two components of thetag from each other. Only the mass tag fragments of the parent ion,which may be referred to as the ‘daughter ions’ are detected in thethird stage of analysis. This allows confirmation that the ion selectedin the first stage of analysis is from a mass marker and not from acontaminating ion, which happens to have the same mass-to-charge ratioas the parent ion. The whole process greatly enhances the signal tonoise ratio of the analysis and improves sensitivity. This mass markerdesign also compresses the mass range over which an array of massmarkers is spread as mass markers can have the same mass as long as theygive rise to mass tag fragments that are uniquely resolvable. Moreover,with isotopes, this mass marker design allows the synthesis of markers,which are chemically identical, have the same mass but which are stillresolvable by mass spectrometry. Use of these markers to identifyoligonucleotide probes is described.

Thus, the prior art provides oligonucleotide probes cleavably linked totags that are detectable by mass spectrometry. The prior art also showsthat these probes enable multiplexing of nucleic acid probe bindingassays. However, multiplexed assays require more than just multipletags. Many nucleic acid probe binding assays do not function well whenmultiplexed because of problems of cross-hybridisation. This is aparticular problem for polymerase chain reaction (PCR) based assays, forwhich it is very costly and time-consuming to optimize reactionsinvolving multiple primer pairs. The problems are due to the high riskof cross hybridization of primers to incorrect templates leading tocross-amplification of templates and hence to incorrect results.

However, some nucleic acid probe binding assay methods that enablehigh-order multiplexing are known in the art. Most notably,Oligonucleotide Ligation Assays (OLA) such as those described in U.S.Pat. No. 4,988,617, which discloses an assay for determining thesequence of a region of a target nucleic acid, which has a knownpossible mutation in at least one nucleotide position in the sequence.In this sort of assay, two oligonucleotide probes that are complementaryto immediately adjacent segments of a target DNA or RNA molecule which,contains the possible mutation(s) near the segment joint, are hybridisedto the target DNA. A ligase is then added to the juxtaposed hybridisedprobes. Assay conditions are selected such that when the targetnucleotide is correctly base paired, the probes will be covalentlyjoined by the ligase, and if not correctly base paired due to amismatching nucleotide(s) near the the segment joint, the probes areincapable of being covalently joined by the ligase. The presence orabsence of ligation is detected as an indication of the sequence of thetarget nucleotide.

Similar assays are disclosed in EP-A-185 494. In this method, however,the formation of a ligation product depends on the capability of twoadjacent probes to hybridize under high stringency conditions ratherthan on the requirement of correct base-pairing in the joint region forthe ligase to function properly as in the above U.S. Pat. No. 4,988,617.Other references relating to ligase-assisted detection are, e.g.,EP-A-330 308, EP-A-324 616, EP-A-473 155, EP-A-336 731, U.S. Pat. No.4,883,750 and U.S. Pat. No. 5,242,794.

Ligation mediated assays have a number of advantages over conventionalhybridization based assays. The reaction is more specific thanhybridization as it requires several independent events to take place togive rise to a signal. Ligation reactions rely on the spatialjuxtaposition of two separate probe sequences on a target sequence, andthis is unlikely to occur in the absence of the appropriate targetmolecule even under non-stringent reaction conditions. This means thatstandardised reaction conditions can be used enabling automation. Inaddition, due to the substrate requirements of ligases, incorrectlyhybridised probes with terminal mismatches at the ligation junction areligated with very poor efficiency. This means that allelic sequencevariants can be distinguished with suitably designed probes. Theligation event creates a unique molecule, not previously present in theassay which enables a variety of useful signal generation systems to beemployed to detect the event. This high specificity makes ligation basedassays easier to multiplex as disclosed in provisional U.S. application20030108913.

Further improvements in stringency and multiplexing can be achievedusing circularising probes. Circularising probes comprise a singleoligonucleotide probe, typically about 70 nucleotides in length orgreater, in which the two probe sequences that are to be ligated to eachother are located at either end of the probe molecule. The probesequences are designed so that when they bind to their target sequence,the two probe sequences are brought into juxtaposition. The probesequences can then be ligated to form a closed circular loop of DNA.Since both probe sequences are linked to each other, when one probesequence binds to its target, binding of the second probe sequence takesplace with rapid kinetics. This ensures that intra-molecular ligation ismuch more likely than inter-molecular ligation reducing cross-ligationof probes to very low levels. In addition, cross-ligated probes arestill linear and it is highly unlikely that two or more probes willcross-ligate to form a circular species. Similarly, mismatched probes,i.e. probes that have bound to a target that does not exactly match theprobe sequence, are unable to ligate and therefore will not becircularized. This all means that correctly reacted probes can bedistinguished from incorrectly reacted probes by the fact that correctlyreacted probes are circular. The ability to resolve correctly matchedprobes means that large numbers of probes can be used simultaneously ina single reaction. The key to using circularizing probes lies in beingable to obtain a signal from circularised probes rather than fromnon-circularised probes and various methods have been disclosed in theprior art to date.

The first disclosure of circularizing probes appears to have been madeby Aono Toshiya in JP 4262799 and JP 4304900. These applications bothdisclose the use of ligation reactions with circularising probes.

Circularisation is detected by the ability of circularized probes toundergo linear Rolling Circle Amplification (RCA). The methodologydisclosed in the above Japanese applications comprises contacting thesample in the presence of a ligase with a probe oligonucleotide.Correctly hybridised probes will be circularized by ligation and willact as a template in a RCA polymerization reaction. A primer, which isat least partially complementary to the circularised probe, togetherwith a strand-displacing nucleic acid polymerase and nucleotidetriphosphates are added to the circularized sequences and a singlestranded nucleic acid is formed which has a tandemly repeated sequencecomplementary to the circularized probe and at least partially to thetemplate. The amplification product is then detected either via alabelled nucleotide triphosphate incorporated in the amplification, orby an added labelled nucleic acid probe capable of hybridizing to theamplification product.

Other methods based on RCA of circularized probes have been disclosed inUS patents U.S. Pat. No. 5,854,033 and related divisions of thisapplication published as U.S. Pat. No. 6,344,329, U.S. Pat. No.6,210,884 and U.S. Pat. No. 6,183,960. The most notable differencebetween the disclosure of these applications and the disclosure of JP4262799 and JP 4304900, is the use of hyper-branching RCA. In thismethod, a second primer that is at least partially complementary to thesingle-stranded product of linear RCA of a circularized probe is addedto the reaction. This results in a further geometric amplification ofthe single stranded product.

Another method for resolving circularized probes from non-circularisedprobes is disclosed in WO 95/22623. The methods disclosed in thisapplication exploit the fact that circularized probes are notsusceptible to degradation by exonucleases while unreacted linear probesare susceptible to degradation. In addition, cyclisation of a probe‘locks’, the probe onto its target, i.e. the probes are resistant tobeing separated from their target. This allows circularized probes to bedistinguished from linear probes by subjecting the probes tonon-hybridising conditions. This approach to the use of circularizingprobes is sometimes referred to as Padlock Probe technology.

Despite the ability of mass tags to enable multiplexing of nucleic acidassays, none of the prior art on mass tags provides methods of analysingnucleic acids using circularising probes. Similarly, none of the priorart on circularising probes provides methods of detecting circularisingprobes suggests using mass spectrometry. It is thus an object of thisinvention to provide methods and reagents to exploit the abilities ofboth mass tags and circularising probes to be used in highly multiplexednucleic acid detection assays.

SUMMARY OF INVENTION

In a first aspect the invention provides a method of detecting a targetnucleic acid comprising

-   -   a) contacting the sample, under hybridizing conditions, with a        probe for said target nucleic acid, wherein said probe comprises        two terminal nucleic acid target recognition sequences that are        complementary to and capable of hybridizing to two neighbouring        regions of the target sequence, and wherein the probe is linked        to a tag that is identifiable by mass spectrometry;    -   b) covalently connecting the ends of the hybridized probe with        each other to form a circularized-probe, which interlocks with        the target strand through catenation;    -   c) cleaving the mass tag from the circularized probe; and    -   d) detecting the mass tag by mass spectrometry.

In a second aspect, the invention comprises a method of detecting thepresence of a target nucleic acid in a sample, which method comprises

-   -   a) contacting the sample, under hybridizing conditions, with a        probe for said target nucleic acid, wherein said probe comprises        two terminal nucleic acid target recognition sequences that are        complementary to and capable of hybridizing to two neighbouring        regions of the target sequence, and wherein the probe comprises        a probe identification sequence;    -   b) covalently connecting the ends of the hybridized probe with        each other to form a circularized-probe, which interlocks with        the target strand through catenation;    -   c) hybridizing a probe detection oligonucleotide to the probe        identification sequence present in the said probe, where the        probe detection oligonucleotide is cleavably linked to a mass        tag;    -   d) cleaving the mass tag from the probe detection        oligonucleotide; and    -   e) detecting the mass tag by mass spectrometry.

In a third aspect, the invention provides a method of detecting thepresence of a target nucleic acid in a sample, which method comprises

-   -   a) contacting the sample, under hybridizing conditions, with a        probe for said target nucleic acid, wherein said probe comprises        two terminal nucleic acid target recognition sequences that are        complementary to and capable of hybridizing to two neighbouring        regions of the target sequence, and wherein the probe further        comprises a probe identification sequence and a pair of primer        binding sequences;    -   b) covalently connecting the ends of the hybridized probe with        each other to form a circularized-probe, which interlocks with        the target strand through catenation;    -   c) cleaving the circularized probe such that the opened probe        has the primer binding sequences oriented to enable polymerase        chain reaction amplification of the probe identification        sequence;    -   d) hybridizing a probe detection oligonucleotide to the probe        identification sequence present in the said probe, where the        probe detection oligonucleotide is cleavably linked to a mass        tag;    -   e) performing a primer extension reaction by providing a primer        capable of hybridizing to the primer binding sequence upstream        of the probe identification sequence and extending said primer        with a polymerase having 5′ exonuclease activity, so as to        cleave the mass tag from the probe detection oligonucleotide;        and    -   f) detecting the mass tag by mass spectrometry.

In a fourth aspect, the invention provides a method of detecting thepresence of a target nucleic acid in a sample, which method comprises

-   -   a) contacting the sample, under hybridizing conditions, with a        probe for said target nucleic acid, wherein said probe comprises        two terminal nucleic acid target recognition sequences that are        complementary to and capable of hybridizing to two neighbouring        regions of the target sequence, and wherein the probe further        comprises a probe identification sequence and a pair of primer        binding sequences;    -   b) covalently connecting the ends of the hybridized probe with        each other to form a circularized-probe, which interlocks with        the target strand through catenation;    -   c) contacting one primer binding sequence with a complementary        primer under conditions for rolling circle replication to occur,        to provide a linear extension product;    -   d) contacting the linear extension product with a primer having        the sequence of the second primer binding sequence, under        conditions to provide for hyper-branching rolling circle        replication;    -   e) hybridizing a probe detection oligonucleotide to the probe        identification sequence present in the said probe, where the        probe detection oligonucleotide is cleavably linked to a mass        tag; and    -   f) detecting the mass tag by mass spectrometry.

The first aspect of the invention set out above relates to a method fordetection of a nucleic acid using a circularising probe in which a masstag is present in the probe. The other aspects of the invention set outabove in contrast use a probe detection sequence in the circularisingprobe, wherein the probe detection sequence is detected with a probeattached to a mass tag.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a directly labelled Circularising Probe according tothe first aspect of this invention. The probe comprises two TargetRecognition Sequences (TRS1 and TRS2; marked as the grey regions) ateither end of the probe. The intermediate sequence is shown in white. Amass tag is shown linked to the probe sequence. In some embodiments,more than 1 mass tag may be linked to a probe of the invention.

FIG. 2 illustrates hybridisation of a circularising probe to its targetnucleic acid. It can be seen that the TRS regions are designed tohybridise in juxtaposition on the target, leaving a small gap, which maybe just a missing phosphodiester linkage or a space of one or morenucleotides.

FIG. 3 illustrates a Circularising Probe according to the second aspectof this invention. The probe comprises two Target Recognition Sequences(TRS1 and TRS2; marked as the grey regions) at either end of the probe.The intermediate sequence is shown in white. A Probe Identificationsequence (marked as the black region) is present in the Intermediateregion (marked as the white region). The Probe Identification sequenceis designed to uniquely identify the probe. In some embodiments, morethan 1 Probe

Identification sequence may be present in a probe of the invention.

FIGS. 4 a and 4 b schematically illustrate the use of a directlylabelled Circularising Probe in a method according to the first aspectof this invention. The details of the method are discussed in detail inthe detailed description that follows.

FIGS. 5 a and 5 b schematically illustrate the use of CircularisingProbes that comprise Probe Identification Sequences in a methodaccording to the second aspect of this invention. The details of themethod are discussed in detail in the detailed description that follows.

FIGS. 6 a, 6 b and 6 c schematically illustrate the use of CircularisingProbes that comprise Probe Identification Sequences and Primer BindingSequences in a method according to the third aspect of this invention.The details of the method are discussed in detail in the detaileddescription that follows.

DETAILED DESCRIPTION OF THE INVENTION

This invention describes reagents, methods and kits that exploitcircularising probes to characterise nucleic acids by mass spectrometry.

Definitions

The term ‘MS/MS’ in the context of mass spectrometers refers to massspectrometers capable of selecting ions, subjecting selected ions toCollision Induced Dissociation (CID) and subjecting the fragment ions tofurther analysis.

The term ‘serial instrument’ refers to mass spectrometers capable ofMS/MS in which mass analysers are organised in series and each step ofthe MS/MS process is performed one after the other in linked massanalysers. Typical serial instruments include triple quadrupole massspectrometers, tandem sector instruments and quadrupole time of flightmass spectrometers.

A Linear Circularising Probe (LCP) is probe sequence where the twotermini of the probe comprise Target Recognition Sequences (TRS) thatare designed to hybridise in juxtaposition on a target nucleic acid. The3′ terminus of the probe preferably comprises a free hydroxyl groupwhile the 5′ hydroxyl group is preferably phosphorylated. These probesare designed so that the TRS portions can be covalently linked to eachother after correct hybridization to their target to form a circularmolecule.

A Closed Circularizing Probe (CCP) is simply a name for an LCP whose TRSregions have been covalently linked to form a circular molecule.

A Probe Identification (PI) sequence is a sequence present in an LCPthat allows the LCP to be identified through hybridisation with anappropriate Probe Detection Sequence that is preferably labelled with aunique mass tag.

A Probe Detection Sequence (PDS) is a labelled probe sequence that is atleast partially complementary to a Probe Identification sequence andthrough hybridisation with a PI sequence it can be used to identify thepresence of an LCP or CCP. Generally, the PDS probes are applied in away that ensures that only CCPs are detected.

A Primer Binding Site (PBS) is a sequence that is present in an LCP orCCP that allows for the binding of a primer oligonucleotide so that theprimer can facilitate replication of the CCP. Primers for rolling circlereplication and for PCR can be used with this invention.

A primer for rolling circle replication is referred to as a RollingCircle Primer (RCP) while a primer for PCR is simply referred to as aPCR primer.

Overview of the Invention

Circularising probes have a number of distinct advantages when comparedto other approaches for SNP analysis and Gene Expression Profiling. Themost widely used technologies at the moment that enables analysis ofboth SNPs and Gene Expression are microarrays and Real Time PCR. Both ofthese technologies have a number of disadvantages. Both thesetechnologies typically require conversion of RNA into cDNA by reversetranscription prior to analysis. For mRNA analysis on microarrays, thistypically requires the presence of a polyadenylation sequence at the 5′end of the mRNA to allow a generic amplification reaction. This meansthat RNA species that are not polyadenylated are difficult to analysewith microarray techniques, such as bacterial or viral RNA. For PCRbased analysis of RNA, the lack of polyadenylation is not such a problembut PCR requires a pair of primers to be designed for each RNA species.Because each primer can potentially cross-hybridise and thuscross-amplify incorrect RNA molecules, PCR primer pairs must have a veryhigh level of specificity. However, even with careful optimisation it isvery difficult to design reactions with more than 20 pairs of specificPCR primers. Circularising probes have the advantage that large numberscan be used simultaneously in a single reaction (Hardenbol et al.,Nature Biotechnology 21(6) pages 673-678, 2003) but can be designed forspecific sequences, rather than relying on polyadenylation makingCircularising probes ideal for analysis of bacterial and viral RNA. Inaddition, the ability to analyse numerous species simultaneously willallow analysis of viral RNA and bacterial simultaneously with human mRNAfor example allowing expression changes in both host and infectiousagent to be analysed simultaneously during studies of infection. The useof mass tags to detect circularisation events, as disclosed in thisinvention, has many advantages, since large arrays of isotopic tags canbe generated. The use of isotopic tags means that accuratequantification is enabled as the relative abundances of isotope tags arean accurate indicator of the levels of the expression products.

In addition, the high specificity of circularising probes and theability to accurately measure expression changes with isotopic mass tagsallows both measurement of expression changes and the presence ofgenetic variation to be performed simultaneously. An example of anapplication of this ability would be viral load monitoring, where it isdesirable to detect not only the total amount of virus, but the amountof each genetic variant. This is of importance in management of HIVtreatment where specific genetic variations correspond to differentforms of drug resistance. To be able to monitor this in a single testwould enable much more effective management of this disease. Similarconsiderations apply to the treatment of cancers which also graduallyevolve drug resistance.

Analysis of gene expression has a number of specific issues. Expressionanalysis typically involves the analysis of RNA species. RNA can beconverted to cDNA by reverse transcription and numerous methods areknown in the art (Wang J. et al., Biotechniques 34(2):394-400, “RNAamplification strategies for cDNA microarray experiments.” 2003;Petalidis L. et al., Nucleic Acids Res. 31(22): e142, “Globalamplification of mRNA by template-switching PCR: linearity andapplication to microarray analysis.” 2003; Baugh L. R. et al., NucleicAcids Res. 29(5):E29, “Quantitative analysis of mRNA amplification by invitro transcription.” 2001). However, it has been shown that targetmediated ligation of LCPs can be performed with RNA targets directly,thus avoiding the need for conversion of RNA to cDNA (Nilsson M. et al.,Nat Biotechnol. 18(7):791-793, “Enhanced detection and distinction ofRNA by enzymatic probe ligation.” 2000). Thus in preferred embodimentsof this invention involving RNA targets, it is preferred that LCPs arecontacted directly with the target RNA molecules.

In preferred embodiments of the second aspect of the invention,correctly ligated CCPs are resolved from unreacted or incorrectlyreacted LCPs by RCR. Target mediated ligation of LCPs to form CCPsinterlocks the CCP with its target. It has been shown that RCR doesstill take place in this constrained environment but at a slightly lowerefficiency than free circles (Kuhn H. et al., Nucleic Acids Res.30(2):574-580, “Rolling-circle amplification under topologicalconstraints.” 2002) so where possible it is desirable to separate theCCPs from their target prior to RCR. When the target species is RNA itis possible to degrade the RNA component of an RNA/DNA duplex usingRNAse H. Thus in embodiments of this invention where RCR is to be used,it may be preferred that prior to RCR, the CCP/RNA duplexes are degradedby contacting them with RNAse H.

Finally, in applications where many thousands of RNA species areanalysed it is preferable that a large library of LCPs is applied in asingle reaction and that a captured library of CCPs is generated fromthe RCR reaction as described above so that the library can be probed atleisure with multiple arrays of mass tagged Probe Detection Sequences.

Linear Circularising Probes

A Linear Circularising Probe (LCP) of all aspects of the presentinvention comprise two Target Recognition Sequences (TRSs) whichhybridise to two neighbouring regions of a target sequence. In theaccompanying Figures, these are designated TRS1 and TRS2. The size ofeach of TRS1 and TRS2 may vary and be independent of each other.Usually, one of the TRSs will be designed to detect an allelic sequence,e.g. a target sequence which may be one of two of more possibilities ata specific nucleotide. This may be designated TRS1, though it will beunderstood that this is an arbitrary designation and TRSl may be at the5′ end or the 3′ end of the LCP. The present invention may be used todetermine which of two or more single nucleotide polymorphisms (SNPs) ispresent in a target sequence, by using a set comprising a mixture of twoor more LCPs, each of which has a TRS1 specific for one SNP and a TRS2which will usually be identical for each member of a set of LCPs.

The length of the TRS1 and position of the allelic nucleotide will beselected to allow the TRS1 which is completely homologous to its targetto hybridise to that target sequence and be ligated to TRS2 whilst aTRS1 of the same set which differs by only a single residue does nothybridise sufficiently to undergo ligation with TRS2 when the target isthat for the former TRS1.

Typically, the TRSs may be between 15 and 25 nucleotides in length each,though shorter lengths, e.g. of from 4 or more nucleotides, are notexcluded. The precise size and composition of the TRSs may be selectedby a person of skill in the art taking into account the specific natureof the target.

After TRS1 and 2 have hybridized to the target molecule and any missingnucleotides between the LCP ends have been filled, the probe ends areconnected to each other, typically by ligation with a ligase, to form acovalently Closed Circularizing Probe (CCP) molecule. Exemplary ligasesare T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, and Thermusthermophilus DNA ligase. Alternative ways of effecting such covalentclosure may, for example, be achieved by use of a catalytic RNA moleculeor by chemical ligation.

By selecting the probe as well as the combined length of any gap fillingnucleotides or oligonucleotides properly, the circular molecule formedwill be wound around and will interlock with the target molecule.Typically, the circularized sequence should be 70 bases or greater for aprobe comprised entirely of nucleotide linkages. Typically, for anucleotide probe a size range of from 70 to 100 nucleotides may be used,e.g. a probe of about 80 or about 90 nucleotides in length.

A probe comprised of non-nucleotide linkages may have different stericlimitations and in this way it may be possible to synthesise shorteroligonucleotide probes. It is sufficient, for the purposes of someaspects of the present invention, that only the actual TRS segmentsconsist of nucleotides or optionally functionally analogous structuresthat can undergo ligation. The remainder of the LCP may have anotherchemical composition, comprising, for example, residues selected frompeptides or proteins, carbohydrates or other natural or syntheticpolymers. Such an intermediate structure of non-nucleotide nature mayeven be preferred with regard to stability and ease ofintroducing-labels or tags, and also since a non-nucleotide intermediatestructure will not exhibit a secondary structure or causemishybridization.

If, however, the probe structure does comprise only nucleic acid, thecombined lengths of the component sequences of each LCP shouldpreferably be such that the strands will leave the double helix on thesame face 10 or a multiple of 10 bases apart, 10 bases representingapproximately one turn of the DNA double helix. Leaving a gap of one ormore nucleotides between TRS 1 and TRS 2 may be advantageous as the gapfilling step can improve specificity of the recognition reaction, but agap is not critical and the method of the invention may be performedwithout it just as effectively, i.e. that TRS 1 and 2 are designed to inimmediate juxtaposition on the target molecule, whereupon the two endscan be directly ligated to circularize the LCP to form a CCP.

Typically oligonucleotides for use as LCPs will be linear polymers ofnucleotides and for many of the embodiments of this invention, this ispreferred. It is however possible to introduce branched structures intonucleic acids, producing Y-shaped and comb-shaped branched structures(see for example Reese C. B. & Song Q., Nucleic Acids Res. 27(13):2672-2681, “A new approach to the synthesis of branched and branched cyclicoligoribonucleotides.” 1999; Horn T. et al., Nucleic Acids Res.25(23):4835-4841, “An improved divergent synthesis of comb-type branchedoligodeoxy-ribonucleotides (bDNA) containing multiple secondarysequences.” 1997; Braich R. S. & Damha M. J., Bioconjug Chem.8(3):370-377, “Regiospecific solid-phase synthesis of branchedoligonucleotides. Effect of vicinal 2′,5′- (or 2′,3′) and3′,5′-phosphodiester linkages on the formation of hairpin DNA.” 1997;Horn T. & Urdea MS., Nucleic Acids Res. 17(17):6959-6967, “Forks andcombs and DNA: the synthesis of branched oligodeoxyribonucleotides.”1989).

Branched oligonucleotides are sometimes used to enable signalamplification without resorting to nucleic acid amplification,particularly comb-oligonucleotides in which a primary sequence specificlinear oligonucleotide is linked to a series of secondaryoligonucleotides (Horn T. et al., Nucleic Acids Res. 25(23):4842-4849,“Chemical synthesis and characterization of branchedoligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleicacid quantification assays.” 1997). Thus in those aspects of the presentinvention in which the LCPs comprise a probe detection sequence, theLCPs may have a primary sequence which comprises the TRS sequences ofthe circularising probe and secondary oligonucleotides branched off theprimary sequence, preferably all comprising an identical sequence, whichact as the probe identification sequence. After circularisation of theprimary sequence and removal of unreacted probes, the circularisedsequence can be probed with mass tagged Probe Detection Sequences. Sincethe comb structure allows multiple Probe Identification sequences to beincorporated into a probe of this invention, this enables signalamplification without requiring amplification of the target sequence orthe probe sequence.

The quantity of covalently circularized probe may be increased byrepeating the cyclizing and dehybridizing steps one or more times.Thereby, multiple allele-specific LCPs will find and be ligated to formCCPs on target molecules. It is worth noting that when these reactionsteps are repeated, it is possible that under appropriate conditions thesame target sequence will mediate closure of multiple LCPs to form CCPsas the CCPs can become threaded on the target molecule. This is becausethe CCPs will move, or wander, to some extent along the target moleculeduring the dehybridizing step, making the target sequence available fora renewed hybridization by a non-circularized probe. If non-hybridisingconditions are to be used to separate CCPs from unreacted andincorrectly ligated LCPs, and if multiple probe hybridization andclosure cycles are to be used, it is, of course, necessary that thetarget molecule is reasonably large and that the target sequence is at asufficient distance from the ends of the target molecule that the CCPsremain linked to the target molecule. For practical purposes, the targetsequence should be at least about 200 base pairs from the nearest enddepending on whether and how the target sequence is bound to a solidphase support. If the target sequence is free in solution, a longerdistance may be required, especially in the case of long-lastingdenaturing washes.

There are a number of advantages to gained by employing LCPs that canform covalently closed circular molecules upon correct hybridization totheir target nucleic acids rather than detecting conventional labelledlinear probes: First, each target requires only a single, syntheticprobe molecule. Second, the ligation reaction provides high specificityof detection, since allelic sequence variants can be distinguished bythe ligase. Third, the circularization of correctly hybridised probesprovides a number of ways by which correctly matched probes can bedistinguished from incorrectly matched probes: CCPs catenate with thetarget sequences, thereby becoming substantially insensitive todenaturants, the ends of the CCP become unavailable to exonucleasedigestion and CCPs can mediate Rolling Circle Replication.

Finally, the simultaneous presence of two terminal probe sequences onone molecule confers kinetic advantages in the hybridization step.

Illustrated in FIG. 1 is a Linear Circularising Probe (LCP) according tothe first aspect of this invention in which the probe is directlyconjugated to a mass tag. The two termini of the probe comprise theTarget Recognition Sequence (TRS) portion of the probe. The 3′ terminusof the probe preferably comprises a free hydroxyl group while the 5′hydroxyl group is preferably phosphorylated. FIG. 2 illustrates the samedirectly labeled probe hybridized to a target nucleic acid sequence,such as a DNA strand, via two TRS end segments of the probe, designatedTRS 1 and TRS 2. TRS 1 and TRS 2 are complementary to two respectivealmost contiguous sequences of the target molecule. A small gap is shownbetween the TRS segments. This gap may simply be a missingphosphodiester linkage or it may comprise a gap of 1 or morenucleotides. If the gap comprises a space of one or more nucleotides, itmay be bridged by a second oligonucleotide probe or it may be filled bypolymerase activity in the presence of the necessary nucleotidetriphosphates.

If the target nucleic acid is sufficiently large, the CCP molecule willremain linked to the target molecule even under conditions that wouldrelease or degrade any hybridized non-cyclized LCPs. This is one way inwhich a circularization reaction produces a selectively detectablespecies, indicating the presence of the target molecule in a sample.Conditions that will denature or degrade a hybridized but non-cyclizedprobe include heat, alkali, guanidine hydrochloride, urea and otherchemical denaturants or exonuclease activity, the latter degrading thefree ends of any unreacted LCPs.

Gap-Filling

As described above the TRS portions of an LCP can be designed tohybridize to a target sequence so that there is a small gap between thetwo TRS termini. This gap may be filled by extending the 3′ TRS using apolymerase and 1 or more nucleotide triphosphates or, if the gap issufficiently large it may be filled by one or more ‘GapOligonucleotides’. The principles and procedures for gap-fillingligation are well known in the art as they are used in the method of‘gap LCR’ (Wiedrnann et al., “PCR Methods and Applications” published byCold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY,pages S51-S64, 1994; Abravaya et al., Nucleic Acids Res., 23(4):675-682,1995; European Patent Application EP0439182, 1991). In the “gap LCR”processes described in these publications, the gap-filling methods areapplied to allow the ligation of two independent nucleic acid probes butthese gap-filling are equally applicable to LCPs.

Hybridisation of LCPs with gaps, followed by gap-filling prior toligation is advantageous as it provides higher stringency as multipleindependent steps have to take place for correct closure of an LCP toform a CCP. Since these steps are unlikely to occur by chance,gap-filling offers a means for enhancing discrimination between closelyrelated target sequences. Gap-filling should be performed with adifferent DNA polymerase from the polymerase used for rolling circlereplication discussed later, and this polymerase will be referred toherein as a gap-filling DNA polymerase. Suitable gap-filling DNApolymerases are discussed in more detail later but in short when theyextend the TRS from the 3′ end of a hybridised LCP, they should notdisplace the hybridised TRS from the 5′ end of the LCP. However, whenthe gap between the two TRS regions of an LCP is only a singlenucleotide, then only the correct expected nucleotide needs to be addedto allow extension of the 3′ TRS to fill the gap. As long as the nextbase is not the same as the missing nucleotide, then most DNApolymerases can be used to fill the gap. This missing base is sometimesreferred to as a “stop base”. The use of “stop bases” in the gap-fillingoperation of LCR is described in European Patent Application EP0439182,for example. The principles of the design of gaps and the ends offlanking probes to be joined, as described in EP0439182, are generallyapplicable to the design of the gap spaces between the ends of the TRSportions of the LCPs of this inventions.

In embodiments of this invention which use rolling circle replication,it is possible for the gap-filling polymerase to interfere with rollingcircle replication. To avoid this, the gap-filling DNA polymerase can beremoved by extraction or inactivated with a neutralizing antibody priorto performing rolling circle replication. Such inactivation is analogousto the use of antibodies for blocking Taq DNA polymerase prior to PCR(Kellogg et al., Biotechniques 16(6): 1134-1137, 1994). More preferably,as shown in FIGS. 5 a and 5 b, after hybridization, gap-filling andligation of LCPs to form CCPs, the CCPs (and any unreacted andincorrectly reacted LCPs) can be captured onto a solid phase support bya tethered oligonucleotide. The capture step can also be performed witha biotinylated oligonucleotide, which can be subsequently captured ontoan avidinated solid support. The gap-filling polymerase can then beremoved by washing the solid support and disposing of the liquid phase.Similarly, if the target sequence is captured onto a solid support,ligation of LCPs to form CCPs will leave the CCPs catenated with thetarget sequence and thus locked onto the solid support. This means thatafter ligation, both the gap-filling polymerase and unreacted LCPs canbe washed away.

Directly Labelled Circularising Probes

FIGS. 1, 2, 4 a and 4 b, schematically show an embodiment of thisinvention in which directly labelled probes are used. In FIGS. 4 a and 4b a method for resolving correctly circularised probes from unreactedprobes is shown. In these figures, the method is shown for two probesthat recognise different alleles of a single target sequence. Eachprobe, designated Linear Circularising Probe 1 and Linear CircularisingProbe 2, is covalently linked to and identified by a unique mass marker.After contacting LCP 1 and LCP 2 with the target sequence, only LCP 1 iscapable of hybridising with the target to form a ligatable complex andso in the presence of ligase only LCP 1 is ligated to from a ClosedCircularized Probe (CCP). The unreacted LCP 2 and any remaining LCP 1can then be degraded by exonuclease activity while CCP 1 is protected byvirtue of being circular. The gene 6 exonuclease of phage T7 provides auseful tool for the elimination of excess LCPs and any unreacted gapoligonucleotides. This exonuclease digests DNA starting from the 5′-endof a double-stranded structure. It has been used successfully for thegeneration of single-stranded DNA after PCR amplification (Holloway etal., Nucleic Acids Res. 21:3905-3906 (1993); Nikiforov et al., PCRMethods and Applications 3:285-291(1994)). If a ‘capture’ sequence isincorporated into the LCP design, the surviving CCP 1 can be capturedonto a solid phase support. The support can then be washed and in thisway exonuclease digested LCP 2 and unreacted LCP 1, which cannothybridise to the solid support, can be separated from the captured CCP1. After washing away LCP 2 and its corresponding tags, the tags on CCP1 can be cleaved from the CCP molecule. If the tags are linked via atrypsin cleavable linkage the tags can be easily cleaved by this enzyme.The solution phase containing the tags can then be injected into a massspectrometer for detection of the tags.

Although only two tags have been shown in the shematic diagram in FIGS.4 a and 4 b, many thousands of different LCPs can be used together ashas been demonstrated previously (Hardenbol et al., Nature Biotechnology21(6) pages 673-678, 2003).

In a further embodiment of this aspect of the invention, shownschematically in FIGS. 7 a and 7 b. Mass Tagged LCPs may be designedwith a cleavable group in them. The cleavable group is positionedbetween the tag and the portion of the LCP that will allow it to becaptured onto the solid support. In FIG. 7 a, it can be seen that acapture sequence is present allowing the LCP to be captured byhybridisation to a tethered or biotinylated oligonucleotide. It wouldalso be possible to directly biotinylate the LCPs. The presence of thecleavable group means that CCPs may be cleaved after their formationfrom LCPs. The cleavage step may take before or after the CCPs arecapture onto a solid phase support. In FIG. 7 b the cleavage is showntaking place before the capture step. The cleavage step ensures that thetagged portion of any unreacted LCPs is not retained on the solidsupport, as the tagged portion of the LCP is only linked to the capturesequence by the cleavable group. The ligation of LCPs to form CCPs meansthat the tag is linked through the ligated portion of the probe so thatafter the cleavage step the mass tags remain linked to the capturesequence (or biotinylated portion) of the probe. In this way, tags willonly be captured for correctly closed CCPs allowing the tags fromunreacted LCPs to be washed away as shown in FIG. 7 b.

The cleavable group may be a type IIS restriction endonucleaserecognition sequence, in which case the capture sequence may also serveas the cleavage site by providing the restriction sequence. In thissituation, the tethered or biotinylated oligonucleotide is preferablyhybridised with the LCPs and CCPs prior to cleavage to form a doublestranded substrate for the restriction endonuclease. Alternatively, thecleavable group my be chemically cleavable. Replacement of one of thephosphodiester linkages in the backbone of an LCP with3′-(N)-phosphoramidate or a 5′-(N)-phosphoramidate, results in a linkagethat is more susceptible to acid hydrolysis than the rest of the probe.Alternatively, a uracil residue can be incorporated into thephosphodiester backbone. This residue is a substrate for the enzymeuracil deglycosylase, which depurinates this residue. The depurinatedresidue is then much more susceptible to hydrolysis than the rest of theprobe molecule.

Indirect Detection of Circularising Probes

In an alternative preferred embodiment of this invention, each differentLCP of this invention comprises a unique Probe Identification (PI)sequence by which it can be identified through hybridisation with anappropriate Probe Detection Sequence that is labelled with a unique masstag.

PI sequences are incorporated in the intermediate region of an LCP. EachPI sequence should uniquely identify its LCP. The PI Sequence isdesigned to allow detection by a corresponding mass tagged ProbeDetection Sequence (PDS). The PI sequences, when amplified duringRolling Circle replication, result in tandemly repeated sequences thatare complementary to the sequence of the mass tagged PDS probes. It maybe desirable to have two or more PI sequences on an LCP as these willincrease the signal from correctly hybridised mass-tagged PDS probes.There is no theoretical limit to the number of PI sequences that can bepresent in an LCP except the practicality of synthesizing and using verylarge LCPs comprising large numbers of PI sequences. When there aremultiple PI sequences, they may have the same sequence or they may havedifferent sequences, with each different sequence complementary to adifferent PDS probe. It is preferred that an LCP contain PI sequencesthat have the same sequence such that they are all complementary to asingle PDS probe. The PI sequences can each be any length that supportsspecific and stable hybridization between the PI sequences and PDSprobes. For practical purposes, a length of 10 to 35 nucleotides ispreferred, with a length of 15 to 25, for example 15 to 20, nucleotideslong being most preferred.

Similarly, the PDS sequences should have a length that is similar to thePI sequences.

In one embodiment, the Probe Detection Sequence may also be a branchedoligonucleotide. For example, the PDS may comprise multiple sequencescomplementary to its Probe Identification sequence, in addition tocomprising a mass tag. Such a PDS may be in the form of a Y-shapedoligonucleotide of a structure described by Suzuki Y. et al. (NucleicAcids Symp Ser. 2000;(44):125-126, “Synthesis and properties of a newtype DNA dendrimer.”) comprising three copies of the PDS. A secondY-shaped branched oligonucleotide comprising three copies of the ProbeIdentification sequence when added to the tripartite PDS probe willassemble a dendrimer in which very large numbers of copies of the PDS,and consequently its associated mass tag will be present. If thetripartite PDS sequence is present in excess, then the dendrimer willhave free PDS sequences available for hybridization to the ProbeIdentification sequences present in correctly circularized CCPs. In thisway a very substantial signal amplification can be achieved withoutamplifying the target nucleic acid or CCPs.

FIGS. 5 a and 5 b illustrate an embodiment of the invention in whichLCPs are identified after closure by the ability of CCPs to beselectively amplified by Rolling Circle Replication. In FIG. 5 a, aschematic of a method of detecting DNA sequence variants is illustratedin which a pair of LCPs that identify different alleles of a DNAsequence are used. The LCPs in this assay are identifiable by theirunique Probe Identification sequences. In FIG. 5 a, a preferredembodiment of the invention is illustrated for a pair of probes thatdetect different variants of a single target molecule. In the firststep, the pair of LCPs are contacted with their target sequence. Onlyone of the LCPs matches the target sequence correctly and hybridises toform a duplex, so that in the next step ligation only occurs at thiscorrectly hybridised duplex converting the LCP into a CCP. This circularsequence is now a substrate for Rolling Circle Replication.

In some embodiments of this aspect of the invention, the unreacted LCPscan be degraded by exonuclease, but this is not shown in FIGS. 5 a and 5b. In the next step, hybridisation of a captured primer with the CCPtakes place to form a CCP/primer duplex. In the next step, polymeraseextends the primer generating a tandem repeated sequence complementaryto the CCP where the tandemly repeated complement is captured on a solidphase support. In alternative embodiments that primer sequence may bebiotinylated rather than linked directly to a bead. In this sort ofembodiment, the biotinylated product of the linear extension of theprimer can then be captured onto an avidinated solid phase support afterthe extension reaction. The captured tandem repeat sequences alsocontain the complement of the Probe Identification (PI) sequencespresent in the LCP sequence. In the final steps of the assay shown inFIG. 5 b, these complements of the PI sequences are probed with masstagged Probe Detection Sequences. Since the targets of the PDS probesare captured on a solid phase support, the correctly hybridised PDSprobes will be captured onto the support by the hybridisation reactionallowing unhybridised PDS probes to be washed away. After washing awayunhybridised PDS probes, the mass tags on the correctly hybridised PDSprobes can be cleaved off for subsequent detection by mass spectrometry.

Captured Libraries

Although only two tags have been shown in the schematic diagram in FIGS.5 a and 5 b, many different LCPs, such as several hundred or even morethan a thousand can be used together as has been demonstrated previously(Hardenbol et al., Nature Biotechnology 21(6) pages 673-678, 2003). Ifmany thousands of probes were used in the assay shown in FIGS. 5 a and 5b, the result of the Rolling Circle Replication step in which thecircularised probes sequences are copied onto beads will generate a‘captured library’ of circularised probes that represents information inthe probed sample. Captured Libraries have a number of advantages. Afterappropriate washing steps the library can be archived for futureanalysis. In addition, the library can be probed multiple times with thesame mass tagged PDS probes to give signal amplification. In someembodiments of this aspect of the invention the captured library isprobed in multiple sequential assays rather than in a single step usingmultiple distinct libraries of mass tagged PDS probes. In this way thesame tags can be used to detect different Probe Identification sequencesin the Captured Library. Thus, the use of Captured Libraries is anespecially preferred embodiment of this invention. For the purposes ofarchiving Captured Libraries, it may be desirable to synthesise thecaptured libraries with exonuclease resistant nucleotide analogues thatare compatible with polymerases such as boranophosphate nuceleotides, oralpha-thio deoxynucleotide triphosphates.

Similarly, for long term storage, it may be preferable to generatecaptured libraries with covalently tethered oligonucleotides rather thanwith biotinylated oligonucleotides that are later captured ontoavidinated beads to avoid the risk of sample loss by dissociation of thenon-covalent biotin/avidin complex.

Rolling Circle Replication

In preferred embodiments of the second aspect of the invention, rollingcircle replication is applied to CCPs generated by target mediatedligation of LCPs. To effect Rolling Circle Replication (RCR) thecircular single-stranded CCP DNA molecules are contacted with RollingCircle Primers (RCPs) that hybridise to Primer Binding Sites in theCCPs. Extension of the RCPs by a strand displacing polymerase willresult in tandem repeats of the complement of the CCP sequence as shownin FIGS. 5 a and 5 b. It can be seen from FIGS. 5 a and 5 b that inpreferred embodiments the RCP is immobilized on a solid phase support orit is capable of being immobilized on a solid support after extensionand Rolling Circle Replication of hybridised CCPs, by using abiotinylated RCP for example.

Specifically FIGS. 5 a and 5 b show a schematic of a method comprisingthe following steps:

(a) mixing one or more Linear Circularising Probes (LCP) with a targetnucleic acid under conditions promoting hybridization, resulting inLCP-target duplexes,

(b) contacting the LCP-target duplexes with a ligase, resulting in aligation mixture, and incubating the ligation mixture under conditionspromoting ligation of the LCPs to form CCPs,

(c) contacting a rolling circle primer (RCP) under conditions thatpromote hybridization with the ligation mixture, resulting in a RCP-CCPduplex,

(d) contacting the RCP-CCP duplex with a DNA polymerase under conditionspromoting extension of the RCP to produce the complement of the CCPsequence, such that continuous extension of the RCP results in formationof tandem repeats of the complement of the CCP sequence.

Although FIGS. 5 a and 5 b show a schematic of an embodiment in whichonly 2 LCPs are present, thousands of LCPs may be present in a singlereaction. Those LCPs that are ligated to form CCPs will be able tosupport RCA and thus will generate captured tandem repeats of theircomplement on a solid support. The solid support bound complementsequences for a number of different CCPs will be referred to as aCaptured CCP Library.

In different embodiments of the second aspect of this invention, theTarget Recognition Sequences may hybridize to the target nucleic acidsequence, with or without a central gap to be filled by one or more gapnucleotides or oligonucleotides.

For the purposes of Rolling Circle Replication (RCR) each LCP shouldcomprise a Primer Binding Sequence (PBS). The PBS is complementary tothe rolling circle primer (RCP). Each LCP should have at least one PBS,although if the LCPs are small, i.e. less than 100 nucleotides in lengththen preferably only a single PBS should be present. This allows rollingcircle replication to initiate at a single site on CCPs. The primercomplement portion and the corresponding rolling circle primer can haveany desired sequence as long as they are complementary to each other. Ingeneral, the sequence of the PBS and the RCP should be chosen so thatthey are not significantly similar to any other portion of the LCP orany LCP in the library, when multiple LCPs are used together. The PBScan be any length that supports specific and stable hybridizationbetween the PBS and the RCP. For this purpose, a length of 10 to 35nucleotides is preferred, with a primer complement portion 16 to 20nucleotides long being most preferred. The PBS can be located anywherewithin the spacer region of an OCP. It is preferred that the PBS isadjacent to the 5′ TRS, with the TRS and the PBS preferably separated bythree to ten nucleotides, and most preferably separated by sixnucleotides.

This location prevents the generation of any other spacer sequences,such as detection tags and secondary target sequences, from unligatedLCPs during DNA replication.

A rolling circle primer (RCP) is an oligonucleotide having sequencecomplementary to the primer binding sequence of an LCP or CCP. Thissequence is referred to as the complementary portion of the RCP. Thecomplementary portion of a RCP and the cognate Primer Binding Sequencecan have any desired sequence so long as they are complementary to eachother. In general, the sequence of the RCP can be chosen such that it isnot significantly complementary to any other portion of the LCP or CCP.The complementary portion of a rolling circle replication primer can beany length that supports specific and stable hybridization between theprimer and the primer complement portion. Generally this is 10 to 35nucleotides long, but is preferably 16 to 20 nucleotides long.

It is preferred that rolling circle replication primers also containadditional sequence at the 5′ end of the RCP that is not complementaryto any part of the LCP or CCP. This sequence is referred to as theDisplacement region of the RCP. The Displacement region is located atthe 5′ end of the primer and serves to facilitate strand displacementduring Rolling Circle Replication. The displacement region is typicallya short sequence, preferably from 4 to 8 nucleotides long, and simplyprovides an unhybridised region of already displaced sequence thatassists the strand displacing polymerase to start displacing theextended RCP.

In some embodiments of the Rolling Circle aspects of this invention,gene 6 exonuclease of phage T7 can be added after the ligation reaction,together with the DNA polymerase to be used to effect Rolling CircleReplication. To protect the Rolling Circle Replication product fromdegradation, the rolling circle primer can be composed of a fewphosphorothioate linkages at the 5′ end, to make the Rolling CirclePrimer and its extension products resistant to the exonuclease(Nikiforov et al. (1994)). The exonuclease will degrade excess LCPmolecules as they can become associated with the rolling circle DNAproduct and interfere with hybridization of PDS probes. The use ofexonucloease digestion is a preferred method of eliminating unreactedLCPs and gap oligonucleotides.

Hyper-Branching Rolling Circle Replication

Contacting a circular template with a single initiating primer and anappropriate polymerase results in linear Rolling Circle Replication andproduces a linear tandemly repeated complementary copy of the circulartemplate. If a second primer is present in the reaction, that iscomplementary to a site in the linear tandemly repeated copy of thecircular template, this will bind to the tandemly repeated sequence atmultiple locations and will initiate further replication. Since thesecond primer will bind at multiple locations, extension that initiatesupstream of a primer will displace the extension product of that primerproviding a linear single stranded template that allows further bindingand extension of the initiating primer. This sort of reaction,therefore, gives rise to geometric amplification of the circulartemplate and is sometimes referred to as hyper-branching RCR and will bereferred to in this way in this application. This is a homogenousgeometric amplification reaction and may be advantageous for use withthis invention. For a fuller discussion on this sort of technique, seeZhang D. Y. et al. (Gene. 274(1-2):209-216, “Detection of rare DNAtargets by isothermal ramification amplification.” 2001) or Lizardi P.M. et al. (Nat Genet. 19(3):225-32. “Mutation detection andsingle-molecule counting using isothermal rolling-circle amplification.”1998).

Accordingly, the use of a hyper-branching RCR reaction may be used inthe present invention in order to provide a means of amplifying theprobe identification sequence following ligation of a LCP.

Microarrays

In further preferred embodiments of this invention, the LCP sequencescomprise a Microarray Address Sequence in the intermediate region of theprobe. A Microarray address sequence will have a sequence that iscomplementary to an oligonucleotide at a specific discrete location on aplanar array.

In embodiments of the invention in which directly labeled LCPs are used,it is possible to hybridise CCPs that form as a result of templatemediated ligation to a microarray. The Microarray Address Sequence willthus ensure that each CCP hybridizes to a discrete location on themicroarray. In this way a combination of distinct Microarray AddressSequences and Mass tags can encode a very large number of LCPs that willthen be uniquely identifiable by a unique combination of theirMicroarray Address Sequence and their Mass Tag. For example 1000discrete Microarray Address Sequences, corresponding to 1000 discretelocations on a microarray, combined with 400 distinguishable Mass Tags,will allow 400 000 different LCPs to be uniquely identified in a singleassay providing an unprecedented level of multiplexing in a singleassay.

In alternative embodiments, in which LCPs are detected through a ProbeIdentification Sequence, which is distinct from the Microarray AddressSequence, the Microarray Address Sequence can be used to ensure thatsubsets of CCPs in a library of CCPs hybridise to distinct locations onthe array. After hybridization, the correctly hybridised microarrayprobe sequence can be extended using an appropriate polymerase to effectrolling circle replication of the hybridised CCPs. Thus, the MicroarrayAddress Sequence is also acting as the binding site for a Rolling CirclePrimer, which happens to be immobilized at a discrete location on aplanar array surface. In this way, a spatially resolved Captured Libraryof CCP sequences can be generated. The captured library can then beprobed by hybridization with PDS sequences that recognize the ProbeIdentification sequence complements generated by the Rolling Circlereplication that takes place at each array location.

After hybridization of directly labeled LCPs or after Rolling CircleReplication and hybridization of PDS sequences to the microarray, themicroarray can then be treated with a MALDI matrix material such as3-hydroxypicolinic acid or alpha-cyano-cinnamic acid. Having preparedthe microarray in this way it can be loaded into a MALDI based massspectrometer and the cleaved tags can be desorbed from discretelocations on the array by application of laser light to the desiredlocation on the array.

In one aspect the invention thus provides a microarray comprising from96 to 1000 discrete locations, such as from 96 to 500 discretelocations, each location comprising a discrete microarray addresssequence complement. In another aspect, the invention provides a kitcomprising such a microarray together with a set of circularising probesof the invention, wherein each member of the set of circularising probescomprises a discrete microarray address sequence which is capable ofhybridizing to a microarray address sequence complement in themicroarray.

In these microarray embodiments of the invention, appropriate methodsfor cleaving the tags from their associated probes on the array must beused. In one preferred approach, the tags are linked to their associatedprobes (linked either directly to LCPs or linked to PDS probes) througha photocleavable linker. This means that cleavage of the tags can takeplace at discrete locations on the array by exposure to light of theappropriate frequency. This light can be applied to the whole arrayprior to analysis by exposing the array to an intense light source.Alternatively, in a MALDI mass spectrometer, the laser used fordesorption can be used to cleave the tags.

In an alternative embodiment, an acid cleavable linker can be used.Since most MALDI matrix materials are acidic, addition of the matrixwill effect cleavage of the mass tags. In a further embodiment, theentire probe label complex can be desorbed, and cleavage of the tags cantake place by collision using Post Source Decay in a Time-Of-Flight massspectrometer or in the mass analyzer of an ion trap instrument or in acollision cell in alternative geometries that are used with MALDI, suchas the Q-TOF geometry.

Practically speaking a microarray could comprise an array of wells onmicrotitre plates, for example, such that each well contains a singleimmobilised oligonucleotide that is a member of the array. In thissituation a sample of the pooled reactions is added to each well andallowed to hybridise to the immobilised oligonucleotide present in thewell. After a predetermined time the unhybridised DNA is washed away.The hybridised DNA can then be melted off the capture oligonucleotide.The released DNA can then be loaded into a capillary electrophoresismass spectrometer or it can be injected into the ion source of a massspectrometer.

Equally, and preferably, the array could be synthesised combinatoriallyon a glass ‘chip’ according to the methodology of Southern or that ofAffymetrix, Santa Clara, Calif. ( see for example: A. C. Pease et al.Proc. Natl. Acad. Sci. USA. 91, 5022-5026, 1994; U. Maskos and E. M.Southern, Nucleic Acids Research 21, 2269-2270, 1993; E. M. Southern etal, Nucleic Acids Research 22, 1368-1373, 1994) or using related ink-jettechnologies such that discrete locations on the glass chip arederivitised with one member of the hybridisation array.

Polymerase Chain Reaction Amplification of CCPs

In another preferred embodiment of this invention, correct closure ofLCPs to form CCPs is detected by Polymerase Chain Reaction. In thisembodiment the LCPs must comprise a pair of PCR Primer Binding Sequences(PPBS). The PPBS sites are preferably oriented so that the first primermust copy across the ligation junction that is formed when the LCP stateof the probe is converted to the CCP by target-mediated ligation. Thismeans that the second PBS site does not become accessible to its primerunless the correct ligation event has taken place.

FIGS. 6 a and 6 b illustrate an embodiment of PCR based assay for thedetection probe circularization using mass tags. These figuresillustrate the assay for a pair of probes but in practice many thousandsof probes could be used simultaneously. In the first stage of the assaythe pair of LCPs are hybridised with their target. Ligation leads toclosure of only one correctly hybridised probe. The probes are capturedonto a solid phase support by an oligonucleotide that also comprises arestriction site for a type II restriction endonuclease. Cleavage of thecaptured probes by the endonuclease results in the formation of a linearstructure in which parts of the LCP sequence have been rearranged. Asimilar process, using uracil deglycosylase to cleave the circularizedprobes, is described by Hardenbol et al. in Nature Biotechnology 21(6)pages 673-678, 2003 and is referred to as ‘molecular inversion’. Thisresults in the PPBS sites being in the correct orientation to enableexponential amplification of the CCPs only in the rearranged probes thathave been correctly ligated by target mediated ligation. In FIG. 6 b,the primer sequences are added along with Mass tagged ProbeIdentification Complement sequences. PCR is then effected with athermostable polymerase with 5′ to 3′ exonuclease activity, which willrelease mass tags from correctly hybridised Probe IdentificationComplement sequences during the PCR reaction as shown in FIG. 6 c. Afterthe PCR reaction the released mass tags can be analysed by massspectrometry.

Target Nucleic Acids

Since the circularising probes, described in the present inventionprovide high specificity, it should be possible to detect the locationof a unique sequence in total vertebrate DNA, particularly Human DNA.Other nucleic acid targets include bacterial DNA, viral DNA and/or RNAand expressed RNA from prokaryotes and eukaryotes.

In addition, the target nucleic acid library to be characterised by themethods and reagents of this invention may, for example, be DNA clonedin an M13 vector, or in a plasmid or phagemid vector that permits theexcision of inserts as circular plasmids. The target nucleic acidmolecule, which may be DNA or RNA and which contains the specificsequence to be detected, should have a sufficient length to ensure thatit can form a double helix, which is required for the circularized probeto interlock or catenate with the target molecule.

The target molecule may be a free molecule, but In some preferredembodiments of this invention, the target nucleic acids may beimmobilized on a solid phase support.

Circularising probes can also be used for ‘in situ’ hybridization totissue slices. With this sort of target, ligation of the LCPs to formCCPs will leave the probes firmly linked to their target sequences, thusallowing extensive washing to be performed. This washing will remove anycircles that may have been formed by non-target-directed ligation, whilecircles ligated on-target are impossible to remove because they aretopologically trapped (Nilsson et al. (1994)).

Reagents

The methods of this invention require a variety of reagents, which arediscussed in detail below.

Oligonucleotide synthesis:

LCPs, gap oligonucleotides, rolling circle primers , PCR primers, masstagged Probe Detection Sequences and any other oligonucleotides can besynthesized using standard oligonucleotide synthesis methods known inthe art. Preferred methods are purely synthetic methods, for example, bythe cyanoethyl phosphoramidite method (Beaucage and Caruthers,Tetrahedron Lett. 22: 1859-1862 (1981); McBride and Caruthers,Tetrahedron Lett. 24: 245-248 (1983)). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). PNA molecules can be made using known methodssuch as those described by Nielsen et al., Bioconjug. Chem. 5:3-7(1994).

Since the circularizing probes of this invention are typically comprisedof a series of distinct sequence components, such as a pair of TRSsequences separated by and intermediate sequence which is common to allprobes, although it may comprise a unique probe identification sequence,it may be desirable to presynthesise these smaller subsequences andassemble them by ligation (Borodina et al., Anal Biochem.318(2):309-313, “Ligation-based synthesis of oligonucleotides with blockstructure.” 2003)

Methods for immobilization of oligonucleotides to solid-phase supportsare well known in the art. For example, suitable attachment methods aredescribed by Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026,1994 and Khrapko et al., Mol Biol (Mosk) (USSR) 25:718-730, 1991. Amethod for immobilization of 3′-amine oligonucleotides on casein-coatedslides is described by Stimpson et al., Proc. Natl. Acad. Sci. USA92:6379-6383 (1995).

Preferred methods of attaching oligonucleotides to solid-statesubstrates are described by Maskos, U. and Southern, E. M., NucleicAcids Res 20(7): 1679-1684, “Oligonucleotide hybridizations on glasssupports: a novel linker for oligonucleotide synthesis and hybridizationproperties of oligonucleotides synthesised in situ”, 1992 and Guo etal., Nucleic Acids Res. 22:5456-5465 (1994).

For many applications of the oligonucleotides of this invention it isuseful to know how stable they are, or more specifically at whattemperature they will dissociate. The stability of DNA duplexes can becalculated using known methods for prediction of melting temperatures(Breslauer, K. J. et al., PNASUSA 83(11): 3746-3750, “Predicting DNAduplex stability from the base sequence.”, 1986; Lesnick and Freier,Biochemistry 34:10807-10815, 1995; McGraw et al., Biotechniques8:674-678, 1990; and Rychlik et al., Nucleic Acids Res. 18:6409-6412,1990).

Mass Tagged Oligonucleotides

A variety of mass tags can be applied with this invention althoughpreferred mass tags are disclosed in WO 97/27327, WO 97/27325, WO97/27331, WO 01/68664 and WO 03/025576. These applications all disclosetags that comprise polyamide compounds, essentially peptides orpeptide-like tags, which means that these tags can be prepared using anumber of peptide synthesis methods that are well known in the art (seefor example Jones J. H., “The chemical synthesis of peptides”, OxfordUniversity Press (1991); Fields G. B. & Noble R. L., Int J Pept ProteinRes 35(3): 161-214, “Solid phase peptide synthesis utilizing9-fluorenylmethoxycarbonyl amino acids.” (1990); Albericio F.,Biopolymers 55(2):123-139, “Orthogonal protecting groups forN(alpha)-amino and C-terminal carboxyl functions in solid-phase peptidesynthesis.” (2000)). In addition, the use of peptide and peptide-liketags enables coupling of these tags to oligonucleotides using a varietyof peptide conjugation techniques that are known in the art.

A preferred mass tag is a tandem mass tag, comprising a mass markermoiety attached via a cleavable linker to a mass normalisation moiety,the mass marker moiety being fragmentation resistant. Such tandem masstags are disclosed in WO01/68664 and WO 03/025576 (which refers to saidtags as “mass labels”), the contents of which are incorporated herein byreference.

Where the present invention is used in the detection of multiplenucleotide sequences using multiple different mass tags, the tandem masstags used may each be a member of a set of related mass tags. Overall,in such a set all of the mass tags in that set will be distinguishablefrom each other by mass spectrometry. This may be achieved by having theaggregate mass of each tag in the set to be same, but each mass markermoiety having a mass different from that of all other mass markermoieties in the set. Alternatively the mass marker moiety can be thesame for each member of the set and the aggregate mass of each member isdifferent from all other tags in that set.

The set of tags need not be limited to the two embodiments describedabove, and may for example comprise tags of both types, provided thatall tags are distinguishable by mass spectrometry, as outlined above.

In one preferred aspect, each mass marker moiety in the set has a commonbasic structure and each mass normalisation moiety in the set has acommon basic structure, and each mass tag in the set comprises one ormore mass adjuster moieties, the mass adjuster moieties being attachedto or situated within the basic structure of the mass marker moietyand/or the basic structure of the mass normalisation moiety. In thisembodiment, every mass marker moiety in the set comprises a differentnumber of mass adjuster moieties and every mass tag in the set has thesame number of mass adjuster moieties.

By “common basic structure”, it is meant that two or more moieties sharea structure which has substantially the same structural skeleton,backbone or core. This skeleton or backbone may be for example compriseone or more amino acids. Preferably the skeleton comprises a number ofamino acids linked by amide bonds. However, other units such as arylether units may also be present. The skeleton or backbone may comprisesubstituents pendent from it, or atomic or isotopic replacements withinit, without changing the common basic structure.

Typically, a set of mass tags of the preferred type referred to abovecomprises mass tags which can be represented by the statement:M(A¹)y-L-X(A²)zwherein M is the mass normalisation moiety, X is the mass marker moiety,A¹ and A² are mass adjuster moieties, L is the cleavable linkercomprising the amide bond, y and z are integers of 0 or greater, and y+zis an integer of 1 or greater. Preferably M is a fragmentation resistantgroup, L is a linker that is susceptible to fragmentation on collisionwith another molecule or atom and X is preferably a pre-ionised,fragmentation resistant group. Preferably M and X have the same basicstructure or core structure, this structure being modified by the massadjuster moieties. The mass adjuster moieties ensure that the sum of themasses of M and X is the same for all mass tags in a set, but that eachX has a distinct (unique) mass.

Mass adjuster moieties may be one or more isotopic substituents situatedwithin the basic structure of the mass marker moiety and/or within thebasic structure of the mass normalisation moiety and/or one or moresubstituent atoms or groups attached to the basic structure of the massmarker moiety and/or attached to the basic structure of the massnormalisation moiety. In a preferred aspect, the mass adjuster moietiesA¹ and A² are independently selected from a halogen atom substituent, amethyl group substituent, a ²H isotopic substituent, a ¹³C isotopicsubstituent or a ¹⁵N isotopic substituent.

In preferred embodiments, the mass tags above are peptides where themass normalisation moiety (M) and the mass marker moiety (X) arecomprised of one or more amino acids, which may be natural amino acidsor modified natural amino acids. In such embodiments, the mass adjustermoieties are isotopic substituents which are present as one or moreatoms of the amino acids.

Preferably the cleavable linker (L) is preferably an amide bond betweenamino acids or may comprise one or more amino acids that facilitatecleavage by collision, such as proline (pro), aspartic acid (asp) or thedipeptide sequence asp-pro.

In a preferred embodiment, neutral amino acids are preferred as a massnormalisation moiety. These may be selected from the group consisting ofalanine, glycine, leucine, phenylalanine, serine, threonine, tryptophanand valine. For the mass marker moiety charged amino acids may be used,since this facilitates ionisation and increases sensitivity. These maybe selected from the group consisting of arginine, asparagine, asparticacid, glutamic acid, glutamine, histidine, lysine and tyrosine.

Preferably a neutral amino acid of the mass normalisation moiety is usedin combination with a charged amino acid of the mass marker moiety.

The mass normalisation and/or mass marker moieties which are amino acidsmay be varied in mass by the mass adjuster moieties as defined above.

Since the preferred compounds for use as mass tags are peptides, it isnecessary to be able to produce peptide/oligonucleotide conjugates toprovide the necessary reagents for this invention. Fortunately, numerousmethods for the preparation of such conjugates are known in the art.There are two general approaches, complete synthesis of the conjugatefor which a number of methods are known (Haralambidis J. et al., NucleicAcids Res. 18(3):493-499, “The synthesis of polyamide-oligonucleotideconjugate molecules.” 1990; de Koning M. C. et al. Curr Opin Chem Biol.7(6):734-740, “Synthetic developments towards PNA-peptide conjugates.”2003) or coupling of independently synthesized peptide oroligonucleotides to each other for which a variety of methods are known.

Methods for coupling tags including peptides to oligonucleotides via 5′amine functionalities are well known in the art (Smith L.M. et al.,Nucleic Acids Res. 13(7):2399-2412, “The synthesis of oligonucleotidescontaining an aliphatic amino group at the 5′ terminus: synthesis offluorescent DNA primers for use in DNA sequence analysis.” 1985; SproatB. S. et al., Nucleic Acids Res. 15(15):6181-6196, “The synthesis ofprotected 5′-amino-2′,5′-dideoxyribonucleoside-3′-O-phosphoramidites;applications of 5′-amino-oligodeoxyribonucleotides.” 1987). In addition,it is possible to incorporate multiple amino groups into anoligonucleotide (Nelson P. S. et al, Nucleic Acids Res.17(18):7179-7186, “A new and versatile reagent for incorporatingmultiple primary aliphatic amines into synthetic oligonucleotides.”1989) to allow multiple tags to be linked to the oligonucleotide.Methods for conjugating peptides to oligonucleotides via thiol groups atthe termini of the oligonucleotides are disclosed in Arar et al.,Bioconjug Chem. 6(5): 573-577, “Synthesis and antiviral activity ofpeptide-oligonucleotide conjugates prepared by using Nalpha-(bromoacetyl)peptides.”, 1995. Oligonucleotides can be coupled topeptides with terminal cysteine residues as disclosed in Wei et al.,Bioconjug Chem. 5(5): 468-74, “Synthesis ofoligoarginine-oligonucleotide conjugates and oligoarginine-bridgedoligonucleotide pairs.”, 1994.

To allow more than one tag to be incorporated per oligonucleotide, masstags can be incorporated into the oligonucleotide through conjugation tothymidine analogues, for example, as disclosed in Brown et al.,Tetrahedron Lett., 42: 2587-2592, “Synthesis of a Modified ThymidineMonomer for Site-Specific Incorporation of Reporter Groups intoOligonucleotides”, 2001. In this publication, a thymidine analogue isdescribed with a linker coupled to the purine ring of the thymidine.This thymidine analogue has a hydroxyl group protected with an FMOCgroup on the end of the linker that can be made available after thenucleotide has been coupled into an oligonucleotide during automatedoligonucleotide syntheisis to allow a phosphoramidite modified tag to beincorporated into an oligonucleotide. Since this analogue can beincorporated within the chain, multiple linkers and hence tags can becoupled to the oligonucleotide.

DNA Ligases

Conversion of LCPs to CCPs is preferably carried out by a DNA ligase .Preferred ligases are those that preferentially form phosphodiesterbonds at nicks in double-stranded DNA. That is, ligases that are unableto ligate the free ends of single-stranded DNA at a significant rate arepreferred. Thermostable ligases are especially preferred. Many suitableligases are known, such as T4 DNA ligase (Davis et al., AdvancedBacterial Genetics—A Manual for Genetic Engineering (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1980)), E. coli DNA ligase(Panasnko et al., J. Biol. Chem. 253:4590-4592 (1978)). In preferredembodiments involving DNA targets, a thermostable DNA ligase is used toeffect closure of LCPs to form CCPs as this will minimize the frequencyof non-target-directed ligation events because ligation takes place athigh temperature (50 to 75 degrees celsius). Examples of thermostableligases include AMPLIGASETM (Kalin et al., Mutat. Res., 283(2):119-123(1992); Winn-Deen et al., Mol Cell Probes (England) 7(3):179-186(1993)), the T. aquaticus DNA ligase (Barany, Proc. Natl. Acad. Sci. USA88: 189-193 (1991), Thermus scotoductus DNA ligase, Rhodothermus marinusDNA ligase and Thermus thermophilus DNA ligase (Thorbjarnardottir etal., Gene 151:177-180 (1995); Housby J. N. et al., Nucleic Acids Res.28(3): E10. (2000)).

The use of a thermostable ligase, enables a wide range of ligationtemperatures to be used, allowing greater freedom in the selection oftarget sequences. A thermostable ligase also makes it easier to selectligation conditions that favor intramolecular ligation. Conditions areeasily found where target mediated ligation of LCPs to form CCPs occursmuch more frequently than tandem linear ligation of two LCPs. Forexample, circular ligation is favored when the temperature at which theligation operation is performed is near the melting temperature (Tm) ofthe least stable of the left target probe portion and the right targetprobe portion when hybridized to the target sequence. When ligation iscarried out near the Tm of the target probe portion with the lowest Tm,the target probe portion is at association/dissociation equilibrium. Atequilibrium, the probability of association in cis (that is, with theother target probe portion of the same LCP) is much higher than theprobability of association in trans (that is, with a different LCP).When possible, it is preferred that the target probe portions bedesigned with melting temperatures near suitable temperatures for theligation operation. T4 DNA ligase is preferred for ligations involvingRNA target sequences due to its ability to ligate DNA ends involved inDNA:RNA hybrids (Hsuih et al., Quantitative detection of HCV RNA usingnovel ligation-dependent polymerase chain reaction, American Associationfor the Study of Liver Diseases (Chicago, Ill., Nov. 3-7, 1995)).

DNA polymerases

DNA polymerases useful in the rolling circle replication step of RCRmust perform rolling circle replication of primed single-strandedcircles. Such polymerases are referred to herein as rolling circle DNApolymerases. For rolling circle replication, it is preferred that a DNApolymerase be capable of displacing the strand complementary to thetemplate strand, termed strand displacement, and lack a 5′ to 3′exonuclease activity. Strand displacement is necessary to result insynthesis of multiple tandem copies of the ligated CCP. Any 5′ to 3′exonuclease activity can result in the destruction of the synthesizedstrand.

It is also preferred that DNA polymerases for use in the disclosedmethod are highly processive. The suitability of a DNA polymerase foruse in the disclosed method can be readily determined by assessing itsability to carry out rolling circle replication. Preferred rollingcircle DNA polymerases are bacteriophage Phi29 DNA polymerase (U.S. Pat.Nos. 5,198,543 and 5,001,050 to Blanco et al.), phage M2 DNA polymerase(Matsumoto et al., Gene 84:247 (1989)), phage PhiPRD1 DNA polymerase(Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)), VENT™ DNApolymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)), Klenowfragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem.45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19(1991)), PRD1 DNA polymerase (Zhu and Ito, Biochim. Biophys. Acta.1219:267-276 (1994)), and T4 DNA polymerase holoenzyme (Kaboord andBenkovic, Curr. Biol. 5:149-157 (1995)).

A further preferred polymerase is the exonuclease(−) BST thermostableDNA polymerase available from New England Biolabs (Mass, USA). BacillusStearothermophilus is a thermophilic bacterium whose polymerase ishighly processive and can be used at elevated temperature (65 degreescentigrade). A Klenow-like fragment without exonuclease activity isavailable (Phang S. M. et al., Gene. 163(1):65-68, “Cloning and completesequence of the DNA polymerase-encoding gene (BstpolI) andcharacterisation of the Klenow-like fragment from Bacillusstearothermophilus.” 1995; Aliotta J. M. et al., Genet Anal.12(5-6):185-195, “Thermostable Bst DNA polymerase I lacks a 3′-->5′proofreading exonuclease activity.” 1996) and it has been shown thatthis polymerase is highly effective for rolling circle replication(Zhang D. Y. et al., Gene. 274(1-2):209-216, “Detection of rare DNAtargets by isothermal ramification amplification.” 2001).

Of these Phi29 DNA polymerase and exo(−) BST DNA polymerase are mostpreferred.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as a helicase. It is considered that any DNApolymerase that can perform rolling circle replication in the presenceof a strand displacement factor is suitable for use in the disclosedmethod, even if the DNA polymerase does not perform rolling circlereplication in the absence of such a factor. Strand displacement factorsuseful in RCA include BMRF1 polymerase accessory subunit (Tsurumi etal., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-bindingprotein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164(1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J.Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad.Sci. USA 91(22):10665-10669 (1994)), single-stranded DNA bindingproteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)),and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635(1992)).

The ability of a polymerase to carry out rolling circle replication canbe determined by using the polymerase in a rolling circle replicationassay such as those described in Fire and Xu, Proc. Natl. Acad. Sci. USA92:4641-4645 (1995).

Another type of DNA polymerase can be used if a gap-filling synthesisstep is used. When using a DNA polymerase to fill gaps, stranddisplacement by the DNA polymerase is undesirable. Such DNA polymerasesare referred to herein as gap-filling DNA polymerases. Unless otherwiseindicated, a DNA polymerase referred to herein without specifying it asa rolling circle DNA polymerase or a gap-filling DNA polymerase, isunderstood to be a rolling circle DNA polymerase and not a gap-fillingDNA polymerase. Preferred gap-filling DNA polymerases are T7 DNApolymerase (Studier et al., Methods Enzymol. 185:60-89 (1990)), DEEPVENT™ DNA polymerase (New England Biolabs, Beverly, Mass.), and T4 DNApolymerase (Kunkel et al., Methods Enzymol. 154:367-382 (1987)). Anespecially preferred type of gap-filling DNA polymerase is the Thermusflavus DNA polymerase (MBR, Milwaukee, Wis.). The most preferredgap-filling DNA polymerase is the Stoffel fragment of Taq DNA polymerase(Lawyer et al., PCR Methods Appl. 2(4):275-287 (1993), King et al., J.Biol. Chem. 269(18):13061-13064 (1994)).

In embodiments of the third aspect of this invention, in which 5′exonuclease activity of the DNA polymerase is used to degrade masstagged PDS probes during PCR amplification of CCPs, it is necessary touse a polymerase with the relevant 5′ exonuclease activity. Taqpolymerase is widely used for this purpose (Livak K. J., Genet Anal.,14(5-6): 143-9, “Allelic discrimination using fluorogenic probes and the5′ nuclease assay.” (1999)) although a variety of polymerases have beenassessed for this purpose and would be applicable with these embodimentsof the invention (Kreuzer K. A. et al., Mol Cell Probes., 14(2): 57-60(2000)).

RNA polymerases

In some embodiments of this invention in which linear Rolling CircleReplication is used an RNA polymerase can be used to effect thereplication reaction. An RNA polymerase which can carry outtranscription in vitro and for which promoter sequences have beenidentified can be used in the disclosed rolling circle replicationmethod. In this sort of embodiment, the Promoter sequences are used asthe Primer Binding Sequences. A DNA primer is required in this sort ofembodiment. The primer must be extended by a non-displacing polymerase,i.e. with the same characteristics as a gap-filling polymerase toproduce a double stranded circular product with a nick. The nick may beligated if desired. The RNA polymerase is then added to the promotersite and will initiate transcription if ribonucleotide triphosphates arepresent. Stable RNA polymerases without complex requirements arepreferred. Most preferred are T7 RNA polymerase (Davanloo et al., Proc.Natl. Acad. Sci. USA 81:2035-2039 (1984)) and SP6 RNA polymerase (Butlerand Chamberlin, J. Biol. Chem. 257:5772-5778 (1982)) which are highlyspecific for particular promoter sequences (Schenborn and Meirendorf,Nucleic Acids Research 13:6223-6236 (1985)). Other RNA polymerases withthis characteristic are also preferred. Because promoter sequences aregenerally recognized by specific RNA polymerases, the OCP or ATC shouldcontain a promoter sequence recognized by the RNA polymerase that isused. Numerous promoter sequences are known and any suitable RNApolymerase having an identified promoter sequence can be used. Promotersequences for RNA polymerases can be identified using establishedtechniques.

Kits:

The LCPs, Gap nucleotides or oligonucleotides, microarrays, Ligases,Polymerases, Primers and Mass Tagged Tag Complement Oligonucleotidesdescribed above can be packaged together in any suitable combination asa kit useful for performing the disclosed methods.

Analysis of Mass tags by Mass Spectrometry:

The essential features of a mass spectrometer are as follows InletSystem ->Ion Source ->Mass Analyser ->Ion Detector ->Data Capture System

There are preferred inlet systems, ion sources and mass analysers andmass analysis methods for the purposes of analysing the mass tags andmass tagged probes of this invention and these are discussed in moredetail below. Inlet systems may comprise separation systems that allowmass tags or mass tagged probes to be separated prior to massspectrometry.

Cleavage of Mass Tagged Oligonucleotides:

The methods of this invention require that mass tags are cleaved fromeither directly labeled probes or from Probe Detection Sequences.Numerous methods are known in the art for the cleavage of probes fromtheir corresponding tags. See for example the disclosures of W098/31830,WO 97/27327, WO 97/27325, WO 97/27331 and WO 98/26095. In preferredembodiments, enzymatic methods may be used. As discussed above, peptideand peptide-like tags are preferred tags for use with the methods ofthis invention. As such, specific endoproteases like trypsin are usefulfor cleaving tags which comprise specific amino acids, arginine orlysine in the case of trypsin. Thus a peptide tag comprising an arginineresidue can be cleaved from its probe sequence by contacting theprobe/tag conjugate with trypsin. Advantageously, arginine also givesrise to intense positive ions. Alternatively, chemical cleavage may beused to release tags. With peptide based tags, incorporation of amethionine residue between the tag peptide and the probe sequence allowsthe probe to be cleaved with cyanogen bromide under acidic conditions.As discussed above, in relation to microarrays, photocleavage is also apreferred method of cleaving tags from their associated probes, forwhich details can be obtained from the disclosure of WO 95/04160, whichdescribes methods of synthesising probes and cleaving said probes. Inanother preferred embodiment of this invention cleavage may take placewithin the mass spectrometer by collision. The amino acid proline andaspartic acid undergo low energy collisions. This means thatincorporation of a proline or aspartic acid residue or both together toform an asp-pro linkage between the tag peptide and the probe sequenceallows the probe to be readily cleaved by low energy collision withoutsubstantial dissociation of the tag peptide. Collision cleavage mustobviously take place after injection of the mass tagged probes or probedetection sequences into the mass spectrometer.

Separation of Mass Tagged oligonucleotides by Chromatography orElectrophoresis:

In further embodiments of the second aspect of this invention, librariesof PDS sequences can be generated in which the PDS comprises additionalelectrophoretic or chromatographic mobility modifying components. Thesemobility modifiers may comprise additional nucleotides or may comprisespecifically designed mobility modifiers (Baron, H. et al., NatureBiotechnology 14: 1279-1282 (1996). The mobility modifiers ensure thatPDS probes that recognise different Probe Identification sequences butwhich carry the same mass tag can be resolved by having differentelution times in an electrophoretic or chromatographic separation. Inthis way a large array of PDS probes can be identified by a uniquecombination of their associated mass tag and the size of their mobilitymodifier. After hybridisation of these mobility modified PDS sequenceswith their corresponding probes, the probes are subjected to anelectrophoretic or chromatographic separation prior to analysis by massspectrometry. This is preferably Capillary Electrophoresis or HighPerformance Liquid Chromatography (HPLC), both of which can be coupleddirectly to a mass spectrometer for in-line analysis of the mass taggedoligonucleotides as they elute from the separation column. A variety ofseparation techniques may be performed by HPLC but reverse phasechromatography is the most widely used method for the separation ofoligonucleotides prior to mass spectrometry. In these embodiments of theinvention, the cleavage of the mass tags from their associated probesmust take place within the mass spectrometer. This cleavage ispreferably effected by collision as discussed above. Collision basedcleavage can be effected in the Electrospray Ion Source throughmanipulation of the Cone Voltage as discussed in more detail below.Alternatively, cleavage can take place in the mass analysis cell of anion trap mass spectrometer or in a collision cell in a tandem massspectrometer.

Inlet Systems:

In some embodiments of this invention a chromatographic orelectrophoretic separation is preferred to reduce the complexity of thesample prior to analysis by mass spectrometry. A variety of massspectrometry techniques are compatible with separation technologiesparticularly capillary zone electrophoresis and High Performance LiquidChromatography (HPLC). The choice of ionisation source is limited tosome extent if a separation is required as ionisation techniques such asMALDI and FAB (discussed below) which ablate material from a solidsurface are less suited to chromatographic separations. For mostpurposes, it has been very costly to link a chromatographic separationin-line with mass spectrometric analysis by one of these techniques.Dynamic FAB and ionisation techniques based on spraying such aselectrospray, thermospray and APCI are all readily compatible within-line chromatographic separations and equipment to perform such liquidchromatography mass spectrometry analysis is commercially available.

Ionisation Techniques:

For many biological mass spectrometry applications so called ‘soft’ionisation techniques are used. These allow large molecules such asproteins and nucleic acids to be ionised essentially intact. The liquidphase techniques allow large biomolecules to enter the mass spectrometerin solutions with mild pH and at low concentrations. A number oftechniques are appropriate for use with this invention including but notlimited to Electrospray Ionisation Mass Spectrometry (ESI-MS), Fast AtomBombardment (FAB), Matrix Assisted Laser Desorption Ionisation MassSpectrometry (MALDI MS) and Atmospheric Pressure Chemical IonisationMass Spectrometry (APCI-MS).

Electrospray Ionisation:

Electrospray Ionisation (ESI) requires that the dilute solution of theanalyte molecule is ‘atomised’ into the spectrometer, i.e. injected as afine spray. The solution is, for example, sprayed from the tip of acharged needle in a stream of dry nitrogen and an electrostatic field.The mechanism of ionisation is not fully understood but is thought towork broadly as follows. In a stream of nitrogen the solvent isevaporated. With a small droplet, this results in concentration of theanalyte molecule. Given that most biomolecules have a net charge thisincreases the electrostatic repulsion of the dissolved molecule. Asevaporation continues this repulsion ultimately becomes greater than thesurface tension of the droplet and the droplet disintegrates intosmaller droplets. This process is sometimes referred to as a ‘Coulombicexplosion’. The electrostatic field helps to further overcome thesurface tension of the droplets and assists in the spraying process. Theevaporation continues from the smaller droplets which, in turn, explodeiteratively until essentially the biomolecules are in the vapour phase,as is all the solvent. This technique is of particular importance in theuse of mass labels in that the technique imparts a relatively smallamount of energy to ions in the ionisation process and the energydistribution within a population tends to fall in a narrower range whencompared with other techniques. The ions are accelerated out of theionisation chamber by the use of electric fields that are set up byappropriately positioned electrodes. The polarity of the fields may bealtered to extract either negative or positive ions. The potentialdifference between these electrodes determines whether positive ornegative ions pass into the mass analyser and also the kinetic energywith which these ions enter the mass spectrometer. This is ofsignificance when considering fragmentation of ions in the massspectrometer. The more energy imparted to a population of ions the morelikely it is that fragmentation will occur through collision of analytemolecules with the bath gas present in the source. By adjusting theelectric field used to accelerate ions from the ionisation chamber it ispossible to control the fragmentation of ions. This is advantageous whenfragmentation of ions is to be used as a means of removing tags from alabeled biomolecule. Electrospray ionisation is particularlyadvantageous as it can be used in-line with liquid chromatography andcapillary electrophoresis, referred to as Liquid Chromatography MassSpectrometry (LC-MS) and Capillary Electrophoresis Mass Spectrometry(CE-MS) respectively.

Atmospheric Pressure Chemical Ionisation:

Atmospheric Pressure Chemical Ionisation (APCI) is similar to (ESI) inthat a dilute solution of the analyte molecule can be ‘atomised’ ornebulised into the ion source at atmospheric pressure, howeverionisation takes place by chemical ionisation. In APCI the ion source isfilled with a bath gas that is subjected to a coronal discharge sourcewhich essentially generates a plasma ionising the bath gas, which inturn ionises the molecules that are sprayed into the ion source. APCIcan also be coupled to laser desorption ionisation (Coon J. J. et al.,Rapid Commun Mass Spectrom., 16(7): 681-685, “Atmospheric pressure laserdesorption/chemical ionization mass spectrometry: a new ionizationmethod based on existing themes.” (2002)), which may also beadvantageous in certain embodiments of this invention, particularly themicroarray embodiments. In general APCI is a relatively mild techniqueappropriate for analysis of mass tags.

Matrix Assisted Laser Desorption lonisation (MALDI):

MALDI requires that the biomolecule solution be embedded in a largemolar excess of a photo-excitable ‘matrix’. The application of laserlight of the appropriate frequency results in the excitation of thematrix which in turn leads to rapid evaporation of the matrix along withits entrapped biomolecule. Proton transfer from the acidic matrix to thebiomolecule gives rise to protonated forms of the biomolecule which canbe detected by positive ion mass spectrometry, particularly byTime-Of-Flight (TOF) mass spectrometry. Negative ion mass spectrometryis also possible by MALDI TOF. This technique imparts a significantquantity of translational energy to ions, but tends not to induceexcessive fragmentation despite this. Accelerating voltages can again beused to control fragmentation in variations of this technique, such asPost Source Decay. The use of laser desorption techniques isparticularly compatible with applications of this invention wheremicroarray are used to analyse CCPs with Microarray Address Sequences.

Fast Atom Bombardment:

Fast Atom Bombardment (FAB) has come to describe a number of techniquesfor vaporising and ionising relatively involatile molecules. In thesetechniques a sample is desorbed from a surface by collision of thesample with a high energy beam of xenon atoms or caesium ions. Thesample is coated onto a surface with a simple matrix, typically a nonvolatile material, e.g. m-nitrobenzyl alcohol (NBA) or glycerol. FABtechniques are also compatible with liquid phase inlet systems—theliquid eluting from a capillary electrophoresis inlet or a high pressureliquid chromatography system pass through a frit, essentially coatingthe surface of the frit with analyte solution which can be ionised fromthe frit surface by atom bombardment.

Mass Analysers:

Fragmentation of peptides by collision induced dissociation is used inthis invention to identify tags on proteins. Various mass analysergeometries may be used to fragment peptides and to determine the mass ofthe fragments. MS/MS and MS^(n) analysis of peptide Tandem Mass Tags:Tandem mass spectrometers allow ions with a pre-determinedmass-to-charge ratio to be selected and fragmented by collision induceddissociation (CID). The fragments can then be detected providingstructural information about the selected ion. When peptides areanalysed by CID in a tandem mass spectrometer, characteristic cleavagepatterns are observed, which allow the sequence of the peptide to bedetermined. Natural peptides typically fragment randomly at the amidebonds of the peptide backbone to give series of ions that arecharacteristic of the peptide. CID fragment series are denoted a_(n),b_(n), c_(n), etc. for cleavage at the n^(th) peptide bond where thecharge of the ion is retained on the N-terminal fragment of the ion.Similarly, fragment series are denoted x_(n), y_(n), z_(n), etc. wherethe charge is retained on the C-terminal fragment of the ion.

Trypsin and thrombin are favoured cleavage agents for tandem massspectrometry as they produce peptides with basic groups at both ends ofthe molecule, i.e. the alpha-amino group at the N-terminus and lysine orarginine side-chains at the C-terminus. This favours the formation ofdoubly charged ions, in which the charged centres are at oppositetermini of the molecule. These doubly charged ions produce bothC-terminal and N-terminal ion series after CID. This assists indetermining the sequence of the peptide. Generally speaking only one ortwo of the possible ion series are observed in the CID spectra of agiven peptide. In low-energy collisions typical of quadrupole basedinstruments the b-series of N-terminal fragments or the y-series ofC-terminal fragments predominate. If doubly charged ions are analysedthen both series are often detected. In general, the y-series ionspredominate over the b-series.

In general peptides fragment via a mechanism that involves protonationof the amide backbone follow by intramolecular nucleophilic attackleading to the formation of a 5-membered oxazolone structure andcleavage of the amide linkage that was protonated (Schlosser A. andLehmann W. D. J. Mass Spectrom. 35: 1382-1390, “Five-membered ringformation in unimolecular reactions of peptides: a key structuralelement controlling low-energy collision induced dissociation”, 2000).FIG. 16 a shows one proposed mechanism by which this sort offragmentation takes place. This mechanism requires a carbonyl group froman amide bond adjacent to a protonated amide on the N-terminal side ofthe protonated amide to carry out the nucleophilic attack. A chargedoxazolonium ion gives rise to b-series ions, while proton transfer fromthe N-terminal fragment to the C-terminal fragment gives rise toy-series ions as shown in FIG. 16 a. This requirement for anappropriately located carbonyl group does not account for cleavage atamide bonds adjacent to the N-terminal amino acid, when the N-terminusis not protected and, in general, b-series ions are not seen for theamide between the N-terminal and second amino acid in a peptide.However, peptides with acetylated N-termini do meet the structuralrequirements of this mechanism and fragmentation can take place at theamide bond immediately after the first amino acid by this mechanism.Peptides with thioacetylated N-termini, will cleave particularly easilyby the oxazolone mechanism as the sulphur atom is more nucleophilic thanan oxygen atom in the same position. Fragmentation of the amide backboneof a peptide can also be modulated by methylation of the backbone.Methylation of an amide nitrogen in a peptide can promote fragmentationof the next amide bond C-terminal to the methylated amide and alsofavours the formation of b-ions. The enhanced fragmentation may bepartly due to the electron donating effect of the methyl groupincreasing the nucleophilicity of the carbonyl group of the methylatedamide, while the enhanced formation of b-ions may be a result of theinability of the oxazolonium ion that forms to transfer protons to theC-terminal fragment as shown in FIG. 16 b. In the context of thisinvention thioacetylation of the N-terminus of a tag peptide can be usedto enhance cleavage of the tag peptide at the next amide bond.Similarly, methylation of the nitrogen atom of an N-terminal acetyl orthioacetyl group will also enhance cleavage of the adjacent amide bond.

The ease of fragmentation of the amide backbone of a polypeptide orpeptide is also significantly modulated by the side chainfunctionalities of the peptide. Thus the sequence of a peptidedetermines where it will fragment most easily. In general it isdifficult to predict which amide bonds will fragment easily in a peptidesequence. This has important consequences for the design of the peptidemass tags of this invention. However, certain observations have beenmade that allow peptide mass tags that fragment at the desired amidebond to be designed. Proline, for example, is known to promotefragmentation at its N-terminal amide bond (Schwartz B. L., Bursey M.M., Biol. Mass Spectrom. 21:92, 1997) as fragmentation at the C-terminalamide gives rise to an energetically unfavourable strained bicyclicoxazolone structure. Aspartic acid also promotes fragmentation at itsN-terminal amide bond. Asp-Pro linkages, however, are particularlylabile in low energy CID analysis (Wysocki V. H. et al., J Mass Spectrom35(12): 1399-1406, “Mobile and localized protons: a framework forunderstanding peptide dissociation.” 2000) and in this situationaspartic acid seems to promote the cleavage of the amide bond on itsC-terminal side. Thus proline, and asp-pro linkages can also be used inthe tag peptides of this invention to promote fragmentation at specifiedlocations within a peptide.

A typical tandem mass spectrometer geometry is a triple quadrupole whichcomprises two quadrupole mass analysers separated by a collisionchamber, also a quadrupole. This collision quadrupole acts as an ionguide between the two mass analyser quadrupoles. A gas can be introducedinto the collision quadrupole to allow collision with the ion streamfrom the first mass analyser. The first mass analyser selects ions onthe basis of their mass/charge ration which pass through the collisioncell where they fragment. The fragment ions are separated and detectedin the third quadrupole. Induced cleavage can be performed in geometriesother than tandem analysers. Ion trap mass spectrometers can promotefragmentation through introduction of a gas into the trap itself withwhich trapped ions will collide. Ion traps generally contain a bath gas,such as helium but addition of neon for example, promotes fragmentation.Similarly photon induced fragmentation could be applied to trapped ions.Another favorable geometry is a Quadrupole/Orthogonal Time of Flighttandem instrument where the high scanning rate of a quadrupole iscoupled to the greater sensitivity of a reflectron TOF mass analyser toidentify the products of fragmentation.

Conventional ‘sector’ instruments are another common geometry used intandem mass spectrometry. A sector mass analyser comprises two separate‘sectors’, an electric sector which focuses an ion beam leaving a sourceinto a stream of ions with the same kinetic energy using electricfields. The magnetic sector separates the ions on the basis of theirmass to generate a spectrum at a detector. For tandem mass spectrometrya two sector mass analyser of this kind can be used where the electricsector provide the first mass analyser stage, the magnetic sectorprovides the second mass analyser, with a collision cell placed betweenthe two sectors. Two complete sector mass analysers separated by acollision cell can also be used for analysis of mass tagged peptides.

Ion Traps:

Ion Trap mass analysers are related to the quadrupole mass analysers.The ion trap generally has a 3 electrode construction—a cylindricalelectrode with ‘cap’ electrodes at each end forming a cavity. Asinusoidal radio frequency potential is applied to the cylindricalelectrode while the cap electrodes are biased with DC or AC potentials.Ions injected into the cavity are constrained to a stable circulartrajectory by the oscillating electric field of the cylindricalelectrode. However, for a given amplitude of the oscillating potential,certain ions will have an unstable trajectory and will be ejected fromthe trap. A sample of ions injected into the trap can be sequentiallyejected from the trap according to their mass/charge ratio by alteringthe oscillating radio frequency potential. The ejected ions can then bedetected allowing a mass spectrum to be produced.

Ion traps are generally operated with a small quantity of a ‘bath gas’,such as helium, present in the ion trap cavity. This increases both theresolution and the sensitivity of the device as the ions entering thetrap are essentially cooled to the ambient temperature of the bath gasthrough collision with the bath gas. Collisions both increase ionisationwhen a sample is introduced into the trap and dampen the amplitude andvelocity of ion trajectories keeping them nearer the centre of the trap.This means that when the oscillating potential is changed, ions whosetrajectories become unstable gain energy more rapidly, relative to thedamped circulating ions and exit the trap in a tighter bunch giving anarrower larger peaks.

Ion traps can mimic tandem mass spectrometer geometries, in fact theycan mimic multiple mass spectrometer geometries allowing complexanalyses of trapped ions. A single mass species from a sample can beretained in a trap, i.e. all other species can be ejected and then theretained species can be carefully excited by super-imposing a secondoscillating frequency on the first. The excited ions will then collidewith the bath gas and will fragment if sufficiently excited. Thefragments can then be analysed further. It is possible to retain afragment ion for further analysis by ejecting other ions and thenexciting the fragment ion to fragment. This process can be repeated foras long as sufficient sample exists to permit further analysis. Itshould be noted that these instruments generally retain a highproportion of fragment ions after induced fragmentation. Theseinstruments and FTICR mass spectrometers (discussed below) represent aform of temporally resolved tandem mass spectrometry rather thanspatially resolved tandem mass spectrometry which is found in linearmass spectrometers.

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR MS):

FTICR mass spectrometry has similar features to ion traps in that asample of ions is retained within a cavity but in FTICR MS the ions aretrapped in a high vacuum chamber by crossed electric and magneticfields. The electric field is generated by a pair of plate electrodesthat form two sides of a box. The box is contained in the field of asuperconducting magnet which in conjunction with the two plates, thetrapping plates, constrain injected ions to a circular trajectorybetween the trapping plates, perpendicular to the applied magneticfield. The ions are excited to larger orbits by applying aradio-frequency pulse to two ‘transmitter plates’ which form two furtheropposing sides of the box. The cycloidal motion of the ions generatecorresponding electric fields in the remaining two opposing sides of thebox which comprise the ‘receiver plates’. The excitation pulses exciteions to larger orbits which decay as the coherent motions of the ions islost through collisions. The corresponding signals detected by thereceiver plates are converted to a mass spectrum by Fourier Transform(FT) analysis.

For induced fragmentation experiments these instruments can perform in asimilar manner to an ion trap—all ions except a single species ofinterest can be ejected from the trap. A collision gas can be introducedinto the trap and fragmentation can be induced. The fragment ions can besubsequently analysed. Generally fragmentation products and bath gascombine to give poor resolution if analysed by FT analysis of signalsdetected by the ‘receiver plates’, however the fragment ions can beejected from the cavity and analysed in a tandem configuration with aquadrupole, for example.

Analysis of TMT Labelled Oligonucleotide Probes by MS/MS:

In preferred embodiments of this invention, the circularised probes areidentified by copying successfully circularised probes onto a solidphase support using Linear Rolling Circle Replication. The capturedmultimeric repeats of the circularised probe sequences are then probedwith oligonucleotides conjugated to Tandem Mass Tags (TMTs).

After cleavage of the TMTs from their oligonucleotides, the TMTs areisolated and injected into the ion source of an appropriate MS/MSinstrument. Typically Electrospray Ionisation (ESI) or AtmosphericPressure Chemical Ionisation (APCI) sources will be used. The tags canthen be detected by selected reaction monitoring with a triplequadrupole for example. Briefly, the first quadrupole of the triplequadrupole is set to let through ions whose mass-to-charge ratiocorresponds to that of the parent tag mass of interest. The selectedparent tag ions are then subjected to collision induced dissociation(CID) in the second quadrupole. Under the sort of conditions used in theanalysis of peptides the ions will fragment mostly at the amide bonds inthe molecule the tag fragment. Although the tags all have the same mass,the terminal portion is different because of differences in thesubstituents on either side of the amide bond. Thus the markers can bedistinguished from each other. The presence of the marker fragmentassociated with a parent ion of a specific mass should identify the tagion and consequently its associated oligonucleotide.

Illustration of the Invention

In order to demonstrate the invention further, the following threeillustrations show how the techniques described herein may be used inthe detection of target sequences.

Protocol 1—Detection of HIV Mutations.

There are about 16 approved drugs (Shafer R. W., Clin Microbiol Rev.15(2):247-277, “Genotypic testing for human immunodeficiency virus type1 drug resistance.” 2002) in use for treatment of human immunodeficiencyvirus type 1 (HIV-1). These drugs belong to three mechanistic classes:protease inhibitors, nucleoside and nucleotide reverse transcriptase(RT) inhibitors, and non-nucleoside RT inhibitors. New drugs based onnovel mechanisms, such as cell entry inhibitors and integrase inhibitorsare under development (Gulick R. M., Clin Microbiol Infect.9(3):186-193, “New antiretroviral drugs.” 2003). The reason for thisproliferation of drugs is due to the ability of HIV-1 to evolveresistance to these drugs. Resistance is caused by mutations in thetarget proteins, which are the protease and RT enzymes for the existingapproved drugs. Drug resistance mutations arise most often in treatedindividuals, as a result of selective drug pressure in the presence ofincompletely suppressed virus replication.

HIV-1 isolates with drug resistance mutations, however, may also betransmitted to newly infected individuals. This means that it isextremely important to be able to detect mutations present in the viruspopulation in a patient. Although the mechanism of mutation is not fullyestablished, it is believed that there is a high natural level ofmutation in the HIV-1 virus and that essentially all possible mutationsare generated in the virus population at some point. Drug therapiesselect for particular resistant variant which gradually becomepredominant. It is this process of change in the predominance ofparticular HIV-1 variant that leads to resistance and failure oftherapy. It is thus essential, not only to be able to identify HIV-1mutations but to accurately quantify their presence.

HIV-1 mutations are, by convention, defined as amino acid substitutionswith reference to a specific sequence referred to as the subtype Bconsensus sequence, which can be obtained from the HumanImmunodeficiency Virus Reverse Transcriptase and Protease SequenceDatabase (Shafer R. W. et al., Nucleic Acids Res. 27(1):348-352, 1999)maintained by Stanford University. There are currently, approximately 80amino acids in the HIV genome in which substitutions are known to resultin drug resistance. At some of these 80 amino acid positions, more thanone amino acid can be substituted into the sequence, meaning that morethan 100 amino acid substitutions need to be detected in an HIV-1 assay,requiring the ability to resolve 180 or more probes for each amino acid.Since each amino acid can be produced by more than one codon, thiscorresponds to the possibility of up to 4 distinct codons for each aminoacid probe at the nucleic acid level. Not all of these codon changesoccur at an appreciable level in vivo and since it is the functionalchange, i.e. the amino acid change that needs to be detected, probes fordifferent codons for the same amino acid could be labeled with the samemass tag or could use the same Probe Identification sequence.

In principle, however, all possible changes could be tested for. Inaddition, certain substitutions are functionally equivalent, leucine andisoleucine are often interchangeable. Similarly, valine is ofteninterchangeable with leucine and isoleucine. This means that LCPs todetect functionally equivalent substitutions could use the same mass tagor the same Probe Identification sequence too.

Bi-allelic LCPs TABLE 1 Components of a two probe set for the detectionof the M184V mutation in the HIV-1 reverse transcriptase gene usinglinear Rolling Circle Replication. Component Probe 1 (Met) Probe 2 (Val)5′ Target ATGTATTGATAGATA ACGTATTGATAGATA Recognition Sequence PrimerATGTTAAGTGACCGGCAG ATGTTAAGTGACCGGCAG Binding CA CA Sequence ProbeGATTTGATTAGATTTGGT AGTAATGTGATTTGATAA Identification AA AG Sequence3′ Target ACATATAAATCATCC ACATATAAATCATCA Recognition Sequence

To illustrate the design of probes for an HIV assay, probes are shown inTable 1 that have been designed to detect the M184V mutation in reversetranscriptase that gives rise to AZT resistance (Shirasaka T. et al.,Proc Natl Acad Sci U S A. 90(2):562-566, “Changes in drug sensitivity ofhuman immunodeficiency virus type 1 during therapy with azidothymidine,dideoxycytidine, and dideoxyinosine: an in vitro comparative study.”1993). These probes are designed for amplification by linear RollingCircle Replication and comprise four components: a 5′ Target RecognitionSequence, a Primer Binding Sequence, a Probe Identification Sequence and3′ Target Recognition Sequence. The complete 70 base sequences of LinearCircularising Probes 1 and 2 are shown below and would be phosphorylatedat the 5′ hydroxyl group: LCP1: 5′-ATGTATTGATAGATAATGTTAAGTGACCGGCAGCAGATTTGATTAGA TTTGGTAAACATATAAATCATCC-3′ (TRS1 andTRS2 in bold, PBS in italics) LCP2:5′-ACGTATTGATAGATAATGTTAAGTGACCGGCAGCAAGTAATGTGATTTGATAAAGACATATAAATCATCA-3′

The two Target Recognition Sequences in Table 1 are each 15 bases inlength. It can also be seen from Table 1 that the same Primer BindingSequence (PBS) is used for both probes and that this PBS will bind to a20-mer primer with the following sequence, which is preferablybiotinylated: Primer: 5′-TGCTGCCGGTCACTTAACAT-3′

Conversely, it can be seen from Table 1 that a different 20-mer ProbeIdentification Sequence is used to identify each probe. The design ofthese sequences is based on the disclosure of Brenner et al. in U.S.Pat. No. 5,846,719, which provides a convenient method for designingsets of oligonucleotide tags which will have a minimal ability tocross-hybridise with each other's target sequences. The correspondingProbe Detection Sequences that are used to detect the ProbeIdentification Sequences will have the same sequence as the ProbeIdentification Sequence as they must bind to the complement of the ProbeIdentification Sequence that will be produced by linear RCR of CCPsformed from LCPs that correctly bind their targets. Thus the ProbeIdentification Sequence in LCPL can be detected after linear RCR by thefollowing Probe Detection Sequence (PDS): PDS1:5′-GATTTGATTAGATTTGGTAA-3′

Similarly, the Probe Identification Sequence in LCP2 can be detectedafter linear RCR by this Probe Detection Sequence: PDS2:5′-AGTAATGTGATTTGATAAAG-3

The PDS sequences are linked to a mass tag, preferably by aphotocleavable linker as disclosed in WO 97/27327 or a collisioncleavable linker as disclosed in WO98/31830. However, it is preferredthat the mass tags comprise a short peptide mass tag as disclosed in WO03/025576.

Redundant LCPs:

Table 1 and probes LCP1 and LCP2 illustrate the basic design of a LinearCircularising Probe to assay for an amino acid substitution in the HIV-1reverse transcriptase gene. However, a number of different nucleic acidchanges can give rise to the same amino acid change. Thus multiple LCPsequences could be necessary to detect all possible variants of asequence. Since all these sequences give rise to the same amino acidchange they could all be identified by the same mass tag or ProbeIdentification Sequence. For the M184V mutation this would result in aset of probes as shown in Table 2 corresponding to the probe sets LCP3and LCP4 shown below. TABLE 2 Components of a two probe set for thedetection of the M184V mutation in the HIV-1 reverse transcriptase geneusing linear Rolling Circle Replication where all possible codons aredetected. Component Probe 3 (Met) Probe 4 (Val) 5′ TargetATGTATTGATAGATA ACGTATTGATAGATA Recognition (1) Sequence CCGTATTGATAGATA(2) TCGTATTGATAGATA (3) GCGTATTGATAGATA (4) Primer ATGTTAAGTGACCGGCAGATGTTAAGTGACCGGCAG Binding CA CA Sequence Probe GATTTGATTAGATTTGGTAGTAATGTGATTTGATAA Identification AA AG Sequence 3′ TargetACATATAAATCATCC ACATATAAATCATCA Recognition (1) Sequence ACATATAAATCATCA(2) ACATATAAATCATCA (3) ACATATAAATCATCA (4)

It can be seen that only one probe is required for the detection of aninternal methionine residue as there is only one codon in the human codefor internal methionine residues. Valine, however can be encoded by fourdifferent codons and so four different probes are need for the detectionof nucleic acid mutations that cause valine to be substituted into aprotein. Thus, the complete 70 base sequences of Linear CircularisingProbes 3 and 4 are shown below and would be phosphorylated at the 5′hydroxyl group: LCP3: 5′-ATGTATTGATAGATAATGTTAAGTGACCGGCAGCAGATTTGATTAGATTTGGTAAACATATAAATCATCC-3′ LCP4:5′-ACGTATTGATAGATAATGTTAAGTGACCGGCAGCAAGTAATGTGATTTGATAAAGACATATAAATCATCA-3′5′-CCGTATTGATAGATAATGTTAAGTGACCGGCAGCAAGTAATGTGATTTGATAAAGACATATAAATCATCA-3′5′-TCGTATTGATAGATAATGTTAAGTGACCGGCAGCAAGTAATGTGATTTGATAAAGACATATAAATCATCA-3′5′-GCGTATTGATAGATAATGTTAAGTGACCGGCAGCAAGTAATGTGATTTGATAAAGACATATAAATCATCA-3′

Since only one codon needs to be tested for methionine LCP3 is the sameas LCP1 but LCP4 comprises four different probes to detect all nucleicacid changes that encode for valine, all identified by the same ProbeIdentification Sequence.

LCPs PCR and Hyper-Branching RCR:

The sequences for LCPs 1 to 4 are all designed for linear Rolling CircleReplication and require only one primer sequence. However, for PCRamplification or for hyper-branching RCR, two primers are required andthe corresponding Primer Binding Sequences must be incorporated into thecorresponding LCPs.

1. A method of detecting the presence of a target nucleic acid in asample, which method comprises a) contacting the sample, underhybridizing conditions, with a probe for said target nucleic acid,wherein said probe comprises two terminal nucleic acid targetrecognition sequences that are complementary to and capable ofhybridizing to two neighbouring regions of the target sequence, andwherein the probe is linked to a tag that is identifiable by massspectrometry; b) covalently connecting the ends of the hybridized probewith each other to form a circularized-probe, which interlocks with thetarget strand through catenation; c) cleaving the mass tag from thecircularized probe; and d) detecting the mass tag by mass spectrometry.2. A method according to claim 1 wherein the two neighbouring regions ofthe target sequence are immediately adjacent to each other.
 3. A methodaccording to claim 1 wherein the two neighbouring regions of the targetsequence are separated by a gap, and wherein covalent connection of thesequences is performed by providing an oligonucleotide capable ofhybridising to the sequence between the neighbouring regions of thetarget sequence, and ligating said oligonucleotide to the terminalnucleic acid recognition sequences.
 4. A method according to claim 1wherein the two neighbouring regions of the target sequence areseparated by a gap, and wherein covalent connection of the sequences isperformed by providing a gap-filling polymerase and one or morenucleotide triphosphates to extend the 3′ terminal nucleic acid targetrecognition sequence of the probe to fill the gap, and ligating theterminal nucleic acid recognition sequences.
 5. A method according toany one of the previous claims, wherein the sample is contacted with twoor more different probes capable of binding different alleles of thetarget sequence, under conditions which a probe complementary for anallele present in the sample will form a circularized probe and a probenot complementary for an allele present in the sample will not form acircularised probe, wherein each probe comprises a different mass tag,and wherein the method includes the step of separating circularizedprobes from non-circularized probes.
 6. A method according to claim 5wherein circularized probe is separated from non-circularized probe bydigesting non-circularized probe with an exonuclease.
 7. A methodaccording to claim 5 wherein the probes are captured onto a solidsupport and cleaved such that only circularized probes retain the taggedportion on the solid support.
 8. A method according to claim 1, whereinthe sample is contacted with two or more sets of probes, each probe setcomprising one or more probes for one or more alleles of a targetsequence.
 9. A method according to claim 8, wherein each probe in a setcomprises a tandem mass tag having a mass tag component and a massnormalization component such that the sum of the masses of the twocomponents are the same for each member of the set.
 10. A method ofdetecting the presence of a target nucleic acid in a sample, whichmethod comprises a) contacting the sample, under hybridizing conditions,with a probe for said target nucleic acid, wherein said probe comprisestwo terminal nucleic acid target recognition sequences that arecomplementary to and capable of hybridizing to two neighbouring regionsof the target sequence, and wherein the probe comprises a probeidentification sequence; b) covalently connecting the ends of thehybridized probe with each other to form a circularized-probe, whichinterlocks with the target strand through catenation; c) hybridizing aprobe detection oligonucleotide to the probe identification sequencepresent in the said probe, where the probe detection oligonucleotide iscleavably linked to a mass tag; d) cleaving the mass tag from the probedetection oligonucleotide; and e) detecting the mass tag by massspectrometry.
 11. A method according to claim 10 wherein the twoneighbouring regions of the target sequence are immediately adjacent toeach other.
 12. A method according to claim 10 wherein the twoneighbouring regions of the target sequence are separated by a gap, andwherein covalent connection of the sequences is performed by providingan oligonucleotide capable of hybridising to the sequence between theneighbouring regions of the target sequence, and ligating saidoligonucleotide to the terminal nucleic acid recognition sequences. 13.A method according to claim 10 wherein the two neighbouring regions ofthe target sequence are separated by a gap, and wherein covalentconnection of the sequences is performed by a providing a gap-fillingpolymerase and one or more nucleotide triphosphates to extend the 3′terminal nucleic acid target recognition sequence of the probe to fillthe gap, and ligating the terminal nucleic acid recognition sequences.14. A method according to claim 10, wherein the sample is contacted withtwo or more different probes capable of binding different alleles of thetarget sequence, under conditions which a probe complementary for anallele present in the sample will form a circularized probe and a probenot complementary for an allele present in the sample will not form acircularised probe, wherein each probe comprises a different probeidentification sequence, and wherein the method includes the step ofseparating circularized probes from non-circularized probes.
 15. Amethod according to claim 14 wherein circularized probe is separatedfrom non-circularized probe by digesting non-circularized probe with anexonuclease.
 16. A method according to claim 14 in which thecircularized probes comprise a primer binding site, and said probes arecontacted with a rolling circle primer under conditions for rollingcircle replication to occur, to provide a linear extension product. 17.A method according to claim 16 wherein said rolling circle primer isattached to a solid support.
 18. A method according to claim 16 whereinsaid rolling circle primer is attached to an affinity ligand that allowsthe replication product to be captured onto a solid support derivatisedwith the corresponding ligand for the affinity ligand.
 19. A methodaccording to claim 16 wherein the probe detection oligonucleotide ishybridized to the probe identification sequence present in the linearextension product.
 20. A method according to claim 10 wherein the sampleis contacted with two or more sets of probes, each probe set comprisingone or more probes for one or more alleles of a target sequence.
 21. Amethod according to claim 20, wherein each probe in a set is detectedwith a probe detection oligonucleotide attached to a tandem mass taghaving a mass tag component and a mass normalization component such thatthe sum of the masses of the two components are the same for each memberof the set.
 22. A method of detecting the presence of a target nucleicacid in a sample, which method comprises a) contacting the sample, underhybridizing conditions, with a probe for said target nucleic acid,wherein said probe comprises two terminal nucleic acid targetrecognition sequences that are complementary to and capable ofhybridizing to two neighbouring regions of the target sequence, andwherein the probe further comprises a probe identification sequence anda pair of primer binding sequences; b) covalently connecting the ends ofthe hybridized probe with each other to form a circularized-probe, whichinterlocks with the target strand through catenation; c) cleaving thecircularized probe such that the opened probe has the primer bindingsequences oriented to enable polymerase chain reaction amplification ofthe probe identification sequence; d) hybridizing a probe detectionoligonucleotide to the probe identification sequence present in the saidprobe, where the probe detection oligonucleotide is cleavably linked toa mass tag; e) performing a primer extension reaction by providing aprimer capable of hybridizing to the primer binding sequence upstream ofthe probe identification sequence and extending said primer with apolymerase having 5′ exonuclease activity, so as to cleave the mass tagfrom the probe detection oligonucleotide; and f) detecting the mass tagby mass spectrometry.
 23. The method of claim 22 wherein the primerextension reaction is part of a polymerase chain reaction.
 24. Themethod of claim 22 wherein the circularized probe is cleaved byhybridizing an oligonucleotide to the probe to form a type IIrestriction endonuclease recognition site, and cleaving the probe with atype II restriction endonuclease which recognises the site formed.
 25. Amethod according to claim 22 wherein the two neighbouring regions of thetarget sequence are immediately adjacent to each other.
 26. A methodaccording to claim 22 wherein the two neighbouring regions of the targetsequence are separated by a gap, and wherein covalent connection of thesequences is performed by providing an oligonucleotide capable ofhybridising to the sequence between the neighbouring regions of thetarget sequence, and ligating said oligonucleotide to the terminalnucleic acid recognition sequences.
 27. A method according to claim 22wherein the two neighbouring regions of the target sequence areseparated by a gap, and wherein covalent connection of the sequences isperformed by providing a gap-filling polymerase and one or morenucleotide triphosphates to extend the 3′ terminal nucleic acid targetrecognition sequence of the probe to fill the gap, and ligating theterminal nucleic acid recognition sequences.
 28. A method according toclaim 22, wherein the sample is contacted with two or more differentprobes capable of binding different alleles of the target sequence,under conditions which a probe complementary for an allele present inthe sample will form a circularized probe and a probe not complementaryfor an allele present in the sample will not form a circularised probe,wherein each probe comprises a different probe identification sequence,and wherein the method includes the step of separating circularizedprobes from non-circularized probes.
 29. A method of detecting thepresence of a target nucleic acid in a sample, which method comprises a)contacting the sample, under hybridizing conditions, with a probe forsaid target nucleic acid, wherein said probe comprises two terminalnucleic acid target recognition sequences that are complementary to andcapable of hybridizing to two neighbouring regions of the targetsequence, and wherein the probe further comprises a probe identificationsequence and a pair of primer binding sequences; b) covalentlyconnecting the ends of the hybridized probe with each other to form acircularized-probe, which interlocks with the target strand throughcatenation; c) contacting one primer binding sequence with acomplementary primer under conditions for rolling circle replication tooccur, to provide a linear extension product; d) contacting the linearextension product with a primer having the sequence of the second primerbinding sequence, under conditions to provide for hyper-branchingrolling circle replication; e) hybridizing a probe detectionoligonucleotide to the probe identification sequence present in the saidprobe, where the probe detection oligonucleotide is cleavably linked toa mass tag; and f) detecting the mass tag by mass spectrometry.
 30. Amethod according to claim 1 wherein the probe further comprises amicroarray address sequence.
 31. A method according to claim 29 whereinprior to detection of the mass tag the probe is hybridized to amicroarray at a location having a nucleotide sequence complementary tothe microarray address sequence of the probe.
 32. A method fordetermining a genetic profile from the genome of an organism, saidmethod comprising: a) providing a microarray which has an array ofmicroarray address sequence complements at discrete locations on saidarray; b) performing the method of claim 30 so as to detect the presenceof one or more mass tags at one or more locations of the microarray; andc) correlating the presence of a mass tag at a location with thepresence of a target sequence in the organism.